How to do desalt reaction by passage through a Micro Bio-spin 6 column?

How to do desalt reaction by passage through a Micro Bio-spin 6 column?

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I want to perform the bisulfite sequencing on rRNA to detect the 5-mC. I followed this protocol

• DNA-free™ DNA Removal Kit (Invitrogen) • Purified RNA, Treat RNA according to manufacturer's instructions.

  1. Adjust DNAse-treated RNA to a volume of 10 μl.

2.Sodium Bisulfite Treatment of RNA EpiTect Kit (Qiagen)

• DNAse-treated RNA preparation • PCR thermal cycler • Micro Bio-spin 6 columns (BioRad) • GlycoBlue coprecipitant (LifeTechnologies) • Cooled microcentrifuge

3.Transfer DNAseI-treated RNA solution into a PCR tube.

  1. Add 42.5 μl freshly made bisulfite solution (EpiTect kit) and mix thoroughly by pipetting.

  2. Add 17.5 μl DNA stabilization solution (EpiTect kit) and mix thoroughly by pipetting.

  3. Place samples in a thermal cycler and run the following program to deaminate RNAs:

• 70 °C: 5 min • 60 °C: 60 min • Repeat steps (i) and (ii) two to six times (depending on target RNA abundance and predicted secondary structures) • 25 °C-hold

7.Desalt reaction by passage through a Micro Bio-spin 6 column according to manufacturer's instructions.

  1. Add 1 volume (70 μl) of 1 M Tris-HCl (pH 9.0) and incubate at 37 °C-30 min for desulfonation.

9.Precipitate RNA by adding sodium acetate (pH 5.2) to 0.3 M, 15 μg GlycoBlue coprecipitant and 3 volumes of 100% ethanol and incubate at − 80 °C for ≥ 1 h.

10.Pellet RNA by centrifugation at 4 °C for ≥ 30 min at top speed.

11.Wash RNA pellet with 75% ethanol.

12.Air-dry ≤ 5 min at room temperature.

  1. Store deaminated RNA pellet until further use at − 80 °C.

In the step-7 I got stuck. i do not know whether do I have to use flowthrough or do i have to elute from the column.


Maybe you can take a look at this. I found it through google when searching for the manual as well.

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Materials and Methods

Sea urchins

Adult purple sea urchins, Strongylocentrotus purpuratus, that were collected from the near shore waters of the Pacific ocean and purchased from Marinus Scientific Inc. (Long Beach, CA) or the Southern California Sea Urchin Co. (Corona del Mar, CA). Animals were maintained as described [16]. Individual animals were identified by placing them in individual, numbered plastic cages that were floated in the aquarium. A minimum of five animals were used throughout the time course experiments.

Marine bacteria isolation and identification

One sea urchin was dissected and coelomocytes, gonad, peristomium, pharynx, and gut tissues were collected, swabbed and streaked onto Marine Broth (MB 3.74% MB power, 0.3% yeast extract, 0.5% proteose peptone [Difco Laboratories]) plates and grown at room temperature (RT) for 2 days. Individual colonies of marine bacteria were re-streaked on separate plates, single colonies were grown in 2–5 ml MB cultures at RT with rotation, and genomic DNA was isolated according to [18]. As a positive control for sequencing, Escherichia coli (LE392 and XL1-Blue), was grown overnight in Luria Bertani (LB) broth at 37°C with rotation. Bacteria were pelleted and the cells were resuspended in 600 μl Tris-NaCl-EDTA-SDS-Urea buffer (TNESU 10 mM Tris pH 7.4, 125 mM NaCl, 10 mM EDTA, 1% (w/v) SDS, 8 M Urea) plus proteinase K (6 μg/μl) and incubated at 37 ° C for 1–2 hr. The solution was extracted with phenol/sevag (50% phenol 48% chloroform, 2% isoamyl alcohol), followed by sevag alone, precipitated with 2.5 M NH4Ac and 1 volume of isopropanol at -70°C, and centrifuged at 17,530 x g for 15 min at 4°C. DNA pellets were washed in ethanol, dried and resuspended in distilled water with RNAse A (ThermoFisher Scientific Inc. 1–2 μg/50 μl). PCR amplification of the 16S ribosomal RNA genes employed the 16Sfor (AGA GTT TGA TCC TGG CTC AG) and 16Srev (ACG GTT ACC TTG TTA CGA CTT) primers in a 50 μl reaction of 0.2 μM each primer, 200 μM deoxynucleotides, 1X company supplied buffer, 2.5 U ExTaq polymerase (Takara Bio Inc.), and 40 ng of bacterial genomic DNA. The PCR program was 95°C for 5 min followed by 25–30 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min 30 sec, and a final extension at 72°C for 7 min. Amplicons were cleaned by the ExoSAP-IT kit (USB Corp.) to remove primers, sequenced with the 16Sfor primer using the BigDye Terminator cycle sequencing kit (Applied Biosystems) and evaluated on a CEQ8000 capillary sequencer (Beckman Coulter, Inc.). Multiple passes of the 16S sequence for each isolate were compared and assembled followed by searches of the non-redundant database at NCBI for matches to known bacterial sequences by BLASTn using Geneious R6 ver. 6.1.8 (Biomatters Ltd). Matches to bacterial 16S sequences are listed in Table 1.

Bacteria selection for immune challenge

Initial immune challenges were conducted with Vibrio diazotrophicus, a marine bacterial species originally isolated from the green sea urchin, Strongylocentrotus droebachiensis (19) and obtained from the American Type Culture Collection (#33466), or were conducted with E. coli. Other species used for immune activation included Gram negative (Vibrio tapetis, isolate C2.1) and Gram positive bacteria (Bacillus sp, isolate P3.1) (Table 1). Overnight cultures of marine bacteria were heat-killed for 30 min at 95°C. To ensure the effectiveness of heat killing, bacteria were plated on MB plates and incubated for 48 hrs at RT to ensure that no colonies appeared and that V. diazotrophicus, which is flagellated and motile [19], showed no visible movement by microscopy over 2 min.

Immune challenge and protein sample collection

Sea urchins were immunologically challenged either by injection of LPS (Sigma-Aldrich Co. 1–2 μg/μl of artificial coelomic fluid [aCF: 10 mM CaCl2, 14 mM KCl, 50 mM MgCl2, 398 mM NaCl, 1.7 mM Na2HCO3, 25 mM Na2SO4 [7]) or with 10 4 bacteria resuspended in 150 μl aCF. Volumes of LPS (1–2 μg/ml wCF) or bacteria (10 4 cells/ml wCF) to inject were estimated by sea urchin wCF volume based on weight as described [20]. Animals were injected a second and third time with 10 6 bacteria per ml of wCF at 24 hr and 48 hr after the initial challenge, or they received up to three injections of LPS (1–2 μg/ml wCF) at 24 hr intervals. wCF was withdrawn from the animals 24 hr after the third injection of heat-killed bacteria or the last LPS injection by inserting a 26 gauge needle through the peristomium, and aspirating 1.5 ml of wCF into a syringe preloaded with an ice chilled cocktail of protease inhibitors (10 mM benzamidine, 1 mM phenylmethanesulfonylfluoride [PMSF], 1x Protease Inhibitor Cocktail [Sigma-Aldrich]) in 50 mM Na2HPO4 pH 7.4 to block protein degradation.

Protein isolation

Ni-Sp185/333 protein isolation by affinity to Ni-His60 resin for 1DE/Western blots initially followed the manufacturer’s instructions (ClonTech Laboratories, Inc.). The percentage of Ni-Sp185/333 proteins of total proteins in samples obtained by this protocol was analyzed by densitometry with a ChemiDoc XRS+ imaging system and associated software (Bio-Rad Laboratories, Inc.) by comparing Sp185/333 + bands on Western blots (see below) to identify the corresponding bands on Coomassie stained gels that were run in parallel.

Ni-Sp185/333 protein isolation was optimized by testing a variety of alternative approaches for cell lysis in addition to modifications to the nickel isolation protocol (S S1 Table S1 Fig). wCF diluted into the protease inhibitor cocktail (see above) was sonicated (amplitude 1–3% Sonic Dismembrator 705 ThermoFisher), in the presence of detergent (1% CHAPS or 1% sarkosyl) to aid in cell lysis, increase protein solubility, prevent protein degradation, and prepare the samples for nickel isolation. Cell fragments were removed by centrifugation (7,000 x g, 30 min, 4°C) and the supernatant was passed through a nickel affinity column (bed volume 200 μl Ni-His60, ClonTech). Binding and elution from nickel columns for Sp185/333 proteins was optimized by modifications to the imidazole concentration in the wash and elution buffers (S1 Protocol, S2 Fig). Unbound proteins were removed with 10 column volumes of wash buffer (10 mM NaCl, 50 mM Na2PO4, 10 mM imidazole), and the remaining bound proteins were eluted with 10 column volumes of elution buffer (10 mM NaCl, 50 mM Na2PO4, 300 mM imidazole). The wash and elution fractions were collected separately for each bed volume, and analyzed by 1DE Western blot (see below) to determine the fractions in which Ni-Sp185/333 proteins were present. These elution fractions were combined and used for further analysis. Traces of imidazole and salts were removed by buffer exchange and sample concentration (Amicron Ultra-4 Centrifugal Devices, 3 kDa MW cutoff EMD Millipore Corp.) against distilled water. Samples were further purified by additional desalting by passage through a Micro Bio-Spin 6 chromatography column (Bio-Rad) into 10 mM Tris pH 7.4 based on the manufacturer’s instructions. Protein samples were dried by speed vacuum centrifugation in preparation for isoelectric focusing.

Two dimensional gel electrophoresis (2DE)

The desalted, dried protein samples were dissolved in 1 ml of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% Immobilized pH Gradient [IPG GE Healthcare Life Sciences]) buffer and stored at -20°C. Methods were optimized by altering protein rehydration and introduction into the IEF strip, voltage employed for IEF, and the pH range of the strips (S1 Protocol, S3 Fig). Immediately before use, 50 mM dithiothreitol (DTT) was added to each sample and incubated at RT for 30 min to reduce thiols. Isoelectric focusing strips (pH 3–10 and pH 7–10, 11 cm, linear, Bio-Rad) were rehydrated with 200 μl of dissolved protein by active rehydration (50 V, 12 hr) in a Protean IEF Cell (Bio-Rad). Constant sample volume was used rather than constant protein concentration because the protein samples included echinochrome [21] that often co-eluted with the Sp185/333 proteins and prevented accurate measurement of protein concentration. The strips were focused to separate the nickel-isolated proteins by differences in pI as previously described [22] and as optimized (S1 Protocol). Following the first dimension, the IEF strips were held at 500 V until samples were separated by MW using 4–15% gradient TGX polyacrylamide gels (Bio-Rad) as described [22] with minor changes. Briefly, focused strips were equilibrated for 30 min in equilibration buffer (50 mM Trizma pre-set crystals, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) plus 1% DTT, followed by alkylation of all cysteines and thiols by incubation in equilibration buffer with 2% iodoacetamide. The equilibrated strip was placed in the top of the TGX polyacrylamide gel and covered with ReadyPrep Overlay Agarose (Bio-Rad). Once set, the gel was run at 200 V, constant voltage, for 45 min to 1 hr at 4°C. Duplicate 2DE gels analyzed by Western blots (see below) were used to demonstrate that processing and storage did not result in artifacts to alter spot positions (S1 Protocol S3 Fig).

Western blots

After the proteins were separated in the second dimension, the gel was electro-blotted to transfer the proteins onto a polyvinylidene fluoride (PVDF) membrane using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) in transfer buffer (25 mM Tris Base pH 8.3, 192 mM glycine, 20% methanol). After transfer, the membrane was rinsed in Tris-NaCl (TN, 25 mM Tris pH 7.4, 0.5 M NaCl) followed by Tris-NaCl-Tween (TNT TN with 0.1% Tween 20), and blocked with Blotto (5% [w/v] powdered milk in TNT) overnight at RT. Blotto was replaced with fresh Blotto containing a cocktail of three anti-Sp185/333 antibodies, which were specific to different conserved regions of the Sp185/333 proteins (Fig 1A) as previously described [9,10]. Each antibody was diluted 1:15,000 for 1DE Western blots or 1:7,500 for 2DE Western blots. After washing in TNT, membranes were incubated with goat anti-rabbit immunoglobulins conjugated with horseradish peroxidase (GaR-Ig-HRP, 1:30,000 in Blotto ThermoFisher) for 1 hr. After washing twice in TNT and once in TN, filters were developed in either Western Lighting ECL reagent (PerkinElmer Inc.) for 1DE Western blots or SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) for 2DE Western blots followed by exposure to X-ray film or evaluated on a ChemiDoc XRS+ (Bio-Rad).

Peptide preparation for mass spectrometry

Following 2DE, gels were stained with BioSafe Coomassie Stain (Bio-Rad). In preparation, gels were soaked in fixing solution (50% methanol, 5% acetic acid) for 30 min, stained with BioSafe Coomassie Stain for 1 hr, and destained in double distilled water at 4°C overnight. Gels were scanned and individual spots chosen for mass spectrometric analysis were cut from the gel and digested with trypsin as previously described [22]. Briefly, multiple cycles of dehydration in 50% acetonitrile followed by rehydration in 50 mM NH4HCO3 was done to remove the SDS and protein stains from the gel pieces. Following the last wash and dehydration cycle, gel pieces were rehydrated in 50 mM NH4HCO3 with 12.5 ng/μl trypsin (sequencing grade) and incubated for 45 min on ice followed by 37°C overnight. Digested peptides were extracted from the gel pieces with 25 mM NH4HCO3, acetonitrile, and 10% formic acid and dried by vacuum centrifugation.

Protein identification: ESI-LTQ-MS/MS

Peptides recovered from digested 2DE gel fragments were analyzed using nanospray electrospray ionization (ESI) LTQ linear iontrap (LTQ) tandem mass spectrometry (MS/MS) (ThermoFisher) as previously described [23] with minor changes. Briefly, peptides were loaded onto a C18 reverse-phase column (3.5 μm, 75 μm x 15 cm ThermoFisher) to concentrate and desalt the samples prior to being separated and eluted. The LTQ performed one full MS scan (300–2,000 m/z) to select the five most intense peaks through dynamic exclusion for MS/MS analysis via collision-induced dissociation with helium. Resulting MS raw files were searched against the NCBI protein database for Strongylocentrotus purpuratus containing 38,417 entries (downloaded May 2015) using Proteome Discoverer software (ThermoFisher) with the following parameters: semi-tryptic peptides, up to two missed cleavages, precursor mass tolerance of 1.5 Da, product ion mass tolerance of 1 Da, possible oxidation of methionine (15.995 Da). The resulting protein matches were filtered with the following parameters: at least two peptides per protein, ΔCN >0.1, Xcorr vs charge state of 1.5 for z = 1, Xcorr = 2 for z = 2, Xcorr = 2.25 for z = 3 and Xcorr = 2.5 for z = 4, and the Proteome Discoverer software Percolator [24,25] Target FDR <1%.

Spot detection

Images of the 2DE Western blots were saved as TIFF files for analysis using Quantity One 1-D and spot identification with PDQuest analysis software (Bio-Rad). Protein density was quantified by dividing each membrane into a 7 x 23 grid of 161 equal sized rectangles that were placed on the identical positions on each individual image of the 2DE Western blots based on pI and MW. The average density of each rectangle was used to determine whether Sp185/333 proteins with varying pI identified in multiple sea urchins differed in prevalence within a given MW region on a blot. Duplicate samples from the same sea urchin were run and analyzed in parallel to ensure that the observed shifts were not artifacts of protein spreading in 2DE that resulted from sample processing (S1 Protocol S3 Fig).


This work was supported by the National Key R&D Program of China (2016YFA0501303 to J.Y.), the National Natural Science Foundation of China (31770885 to J.Y.), the Beijing Nova Program (Z171100001117014 to J.Y.) and the US National Institutes of Health (R01 GM102187 and R01 CA174864 to K.S.C. and R01 GM072866 to W.T.L.). This work was also supported by the Wake Forest Baptist Comprehensive Cancer Center (P30CA012197 to W.T.L.). We thank Q. Zhou and W. Leng (National Center for Protein Sciences–Beijing) for expert technical assistance, C. Liu and H. Chi (Institute of Computing Technology, CAS) for helpful discussions in proteomic informatics, K. Tallman and N. Porter (Vanderbilt University) for providing light and heavy Az–UV–biotin reagents, P. Wu (The Scripps Research Institute) for providing the BTTP click ligand, M. Wilson (University of Nebraska, Lincoln) for providing recombinant DJ-1, and S. G. Rhee (Yonsei University College of Medicine) and M. Toledano (Institut des Science du Vivant Frédérique Joliot) for providing Srx +/+ and Srx –/– MEFs.

Materials and Methods

Sample Preparation.

Fluorescein-labeled human eIF3j, the human 43S PIC components (eIF1, eIF1A, eIF2, eIF3, eIF5, 40S subunit, and initiator Met-tRNAi), and wild-type and truncated eIF4GI, eIF4AI, eIF4B, and eIF4E were prepared as described previously (12, 21). The 32-nt and 42-nt CAA repeat mRNAs [5′-GGACAACAACAACAAACC(AUG/CUC)GAACAACAACA ACAACAACAA -3′, where the underlined sequence is specific to the 42-nt mRNAs] were transcribed using T7 RNA polymerase and a synthetic oligo ssDNA template annealed to a T7 promoter strand as described previously (21). The Globin-Luc mRNA and its UTR sequence were transcribed in a similar manner, but using a PCR-amplified DNA as a transcription template. The DNA templates were PCR-amplified from a pUC19 plasmid harboring the T7 promoter, human globin 5′ UTR, followed by a NanoLuc luciferase coding region between the KpnI and HindIII restriction sites, using M13 forward and M13 reverse primers for the Globin-Luc mRNA, or M13 forward and a synthetic reverse primer (5′-GAAATCTTCGAGTGTGAAGACCAT-3′) for the globin UTR sequence. Therefore, the resulting Globin-Luc mRNA included a 160-bp 3′ UTR originating from the sequence between the HindIII and M13 reverse primer sites in the pUC19 backbone. The globin 5′ UTR sequence included the first 24 nt of the NanoLuc coding sequence directly following the globin UTR. Unless indicated otherwise, all mRNAs used in this study were capped with vaccinia capping enzyme (New England BioLabs) according to the manufacturer’s protocol, and then purified with phenol/chloroform extraction, followed by a passage through a Micro Bio-Spin 6 column (Bio-Rad).

For fluorescent labeling, the 3′-end of CAA(AUG)-42 was labeled as described previously (30), using 0.5 mM fluorescein-5-thiosemicarbazide as the reactant. The reaction yielded >95% labeling efficiency as judged by the absorbance spectrum. The labeled RNA was phenol/chloroform-extracted and purified with a Micro Bio-Spin 6 column. Note that the labeling reaction was performed before the capping reaction to eliminate the possibility of the undesired labeling at the 3′ position of m 7 G cap nucleotide.

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1.8</li> <li>For pure RNA 260 nm/230 nm =



Cytosine methylation (formation of 5-methylcytosine, 5mC) is a well-established epigenetic mechanism that affects global gene expression 1,2 . The 5mC remodeling of DNA is used extensively during mammalian development and cell differentiation, as well as during cancer initiation, progression and in the therapeutic response 3,4 . Active demethylation in the mammalian genome is mediated by the TET (Ten-Eleven Translocation) family of dioxygenases that oxidize the 5mC modification to 5-hydroxymethylcytosine (5hmC) 5,6 , and further to 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) 7,8,9 . The “intermediate” 5hmC not only marks active demethylation but also serves as a relatively stable DNA mark that plays distinct epigenetic roles 2,10,11,12,13,14,15 . Recent genome-wide sequencing maps of 5hmC in various mammalian cells and tissues support its role as a marker for gene expression 16,17,18,19,20,21,22 it is enriched in enhancers, gene-bodies and promoters, and changes in 5hmC correlate with changes in gene expression levels 22,23 .

The discovery of cell-free DNA (cfDNA) originating from different tissues in the circulating blood has revolutionary potential for the clinic 24 . Liquid biopsy-based biomarkers and detection tools offer substantial advantages over existing diagnostic and prognostic methods, including being minimally invasive. They thus have a cost-efficient potential to promote higher patient compliance and clinical convenience to enable dynamic monitoring 25 . Tumor-related somatic mutations in cfDNA have been shown to be shared with the tumor tissue, although low mutation frequency and the lack of information on tissue of origin hamper the detection sensitivity. 5mC and 5hmC in cfDNA from liquid biopsies could serve as parallel or more valuable biomarkers for non-invasive diagnosis and prognosis of human diseases, because they recapitulate gene expression changes in relevant cell states. If these cytosine modification patterns can be sensitively detected, disease-specific biomarkers could be identified for effective early detection, diagnosis and prognosis.

High-throughput sequencing is an ideal platform for detecting genome-wide cytosine modification patterns. Whole-genome bisulfite sequencing or alternative reduced representative methods have been applied in biomarker research with cfDNA 26,27,28 . Tissue- and cancer- specific methylation sites have shown promising performance in tracking tissue-of-origin from circulating blood 26,28 . However, 5mC serves mostly as a repressive mark with a high background level in the human genome, and its sequencing with bisulfite treatment has been hampered with extensive DNA degradation, in particular with cfDNA. Taking advantage of the presence of the hydroxymethyl group, selective chemical labeling can be applied to map 5hmC using low levels of DNA with high sensitivity. The profiling method is robust and cost-effective for studies of large cohorts and practical applications. Here, we have established 5hmC-Seal technology for 5hmC profiling in cfDNA. We show that the differentially enriched 5hmC regions in cfDNA are excellent markers for solid tumors.

Materials and Methods

Expression and Purification of Recombinant bMsrA.

An N-terminal poly(His)-tagged form of bMsrA was obtained by overexpression in E. coli. The expression vector was constructed by amplifying the bMsrA gene (17) with PCR mixtures containing Deep Vent DNA Polymerase (New England Biolabs). The flanking restriction sites, NdeI and XhoI, were added with the following primers: 5′ end primer, 5′-G GAA TTC CAT ATG CTC TCG GTC ACC CGT CGT GCC CTC CAG C-3′ 3′ end primer, 5′-ATC TTA CTC GAG TTA CTT TTT AAT ACC C-3′. In addition, the tandem rare Arg codons (AGG) that code for residues 6 and 7 of the protein were replaced by CGU, preferred by E. coli (19). The PCR product was cloned into the Novagen pet28b expression vector, and the sequence was verified. Overexpression of bMsrA from pet28b resulted in the addition of a His-tag to the N terminus of the protein, i.e., GSSHHHHHHSSGLVPRGSH-. Cys to Ser mutants were made by using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) with the following primers and their complement: Cys-72 → Ser, 5′-GCT GTA TTT GGA ATG GGC TCT TTC TGG GGA GGC-3′ Cys-107 → Ser, 5′-CCC AAT CCT ACT TAT AGA GAA GTC TCC TCA GGA AAA ACT GG-3′ Cys-218 → Ser, 5′-CGA CGG GTA CTC CGG CCT CGG GG-3′ and Cys-227 → Ser, 5′-GCA CCG GAG TGT CTT CTC CCC TGG GTA-3′.

Four-liter fermentation cultures of Epicurian Coli BL21-Gold(DE3) cells (Stratagene) containing the expression plasmid were grown in LB-kanamycin broth (100 mg/liter) at 37°C, 750 rpm, and 10–15 liters of air per min. Expression was induced by the addition of isopropyl β- d -thiogalactoside (IPTG) to 0.1 mM at 1.0 OD600 and continuing growth for 4 h at 25°C and 250 rpm. The cells (15–30 g) were suspended in 100 ml of +T/G buffer [50 mM Hepes (pH 7.9)/10% glycerol/0.1% Triton X-100/0.5 M KCl/40 μg/ml DNase/1 mM MgCl2/15 mM methionine/5 mM imidazole/2 Complete/EDTA-free (Boehringer-Mannheim) inhibitor tablets] and lysed by passage through a French press cell. The resulting slurry was centrifuged at 40,000 × g for 30 min. The supernatant was loaded onto a 10-ml NTA-agarose column (Qiagen, Valencia, CA) equilibrated with +T/G buffer at 4°C. After washing with +T/G and −T/G buffer (+T/G buffer without glycerol, Triton X-100, and inhibitor mixture), bMsrA was eluted with a 500-ml gradient from 5 to 250 mM imidazole in −T/G buffer. The fractions corresponding to bMsrA, determined by SDS/PAGE analysis, were pooled. EDTA and DTT were added to give a final concentration of 5 mM and 15 mM, respectively. The protein solution was incubated on ice for 1 h and then dialyzed against 4 liters of 20 mM Mes (pH 6.5)/20 mM NaCl/0.1 mM EDTA (buffer A) at 4°C. The dialyzed protein was loaded in several batches onto a POROS SP/M column (BioCAD system PerSeptive Biosystems, Foster City, CA) equilibrated with 20 mM Mes (pH 6.5)/0.3 M NaCl/0.1 mM EDTA. Protein was eluted at 10 ml/min with a 10-column volume gradient from 0.3 to 0.7 M NaCl. After dialysis overnight against 4 liters of buffer A, the protein was concentrated with an Amicon Centriprep device (10-kDa cutoff). Protein concentrations were determined by absorption at 280 nm with the theoretical extinction coefficient of each variant calculated by using the expasy protparam tool [, Swiss Institute of Bioinformatics wild-type, 31,390 M -1 ⋅cm -1 single and double mutants, 31,270 M -1 ⋅cm -1 triple mutants, 31,150 M -1 ⋅cm -1 ]. The protein concentrations were in good agreement with those values obtained by using the Bradford method of protein determination. Typical yields were 5–25 mg/liter culture.

Analysis of the recombinant E. coli and bovine MsrAs by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and electronic absorption spectroscopy showed no indication that either enzyme contains bound metals or cofactors (data not shown).

MsrA Activity Assay.

The enzymatic activity of bMsrA (2–4 μg) was assayed at 37°C in 25 mM Tris (pH 7.4) essentially as previously described by using N-acetyl- l -[ 3 H]methionine sulfoxide as substrate (165 μM) in a total reaction volume of 30 μl (20). The incubation contained either 15 mM DTT or a thioredoxin-regenerating system (180 pmol of E. coli thioredoxin, 3 μg of E. coli thioredoxin reductase, and 50 nmol of NADPH). The reactions were stopped after 10 min by the addition of 0.5 M HCl (1 ml). The acidified solution was extracted with 3 ml of ethyl acetate, and the amount of product, N-Ac- l -[ 3 H]Met, was determined by quantitating the amount of radioactivity in the ethyl acetate phase, corrected for the efficiency of extraction (50%).

The oligomeric state of the bMsrA variants [2.5 mg/ml, 25 mM Tris (pH 7.4)] was determined either in the absence or in the presence of 0.9 mM Met(O) (10-fold molar excess) without the addition of an exogenous reducing system. After incubation for 1 h at 37°C, sample loading buffer [62.5 mM Tris (pH 6.8)/2% SDS/0.01% bromophenol blue] with and without DTT (100 mM) was added. The samples were heated at 100°C for 10 min and analyzed by SDS/PAGE using 10–15% gradient gels on a Pharmacia PhastSystem.

Mass Spectroscopic Determination of Free Thiols.

The number of free sulfhydryl groups of monomeric bMsrA was determined by derivatization with methyl methanethiosulfonate (MMTS, Sigma-Aldrich, ref. 21) and delayed-extraction matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (PerSeptive Biosystems Voyager-DE Biospectrometry Workstation). Stock enzyme solutions were diluted just before analysis to 100 μM in 25 mM Tris (pH 7.4) and 15 mM DTT and incubated for 10 min at 37°C. DTT was removed by using Micro Bio-Spin 6 columns (Bio-Rad). The enzyme was then incubated with 1 mM of either l -Met(O) or l -Met in 25 mM Tris (pH 7.4) for 1 h at 37°C. Substrate and product were removed by spin columns. The sample was then split into three parts for mass analysis: (i) no further treatment, (ii) addition of MMTS [10 mM Tris (pH 8.0), 233 μM MMTS (4.5 equivalents)] and incubation on ice for 30 min, or (iii) the addition of 10 mM Tris (pH 8.0) and 15 mM DTT and incubation at 37°C for 15 min. The DTT-treated samples were subsequently passed through spin columns to remove DTT and treated with MMTS as described above. The samples were purified and desalted for mass analysis by adding 1 μl of 25% trifluoroacetic acid (TFA) and passing the protein over miniature C18 columns (ZipTip, Millipore) using 0.1% TFA and 50% acetonitrile as wash and elution buffers, respectively. The samples were spotted onto the sample plate in the following manner: 0.5 μl of internal standards [0.1 pmol/μl in 10 mg/ml 3,5-dimethoxy-4-hydroxycinnamic acid (sinnapinic acid, Sigma-Aldrich), 30% acetonitrile, 0.1% TFA E. coli thioredoxin (11,674 Da), horse apomyoglobin (16,952 Da) (PerSeptive Biosystems Sequazyme Kit Mix 3)], 0.5 μl of sample, and 0.5 μl of sinnapinic acid stock. Because of the inherent formation of higher-order oligomers during the evaporation of the samples, only the mass of the monomeric form of each bMsrA variant was used in subsequent comparisons.

Supporting information

S1 Fig. Metabolic profiles of NSUN2-expressing and -lacking cells.

(A) Schematic representation of stem cell differentiation in the absence or presence of NSUN2. (B) Marker expression and cellular differences in hair follicles expressing or lacking NSUN2. (C) Gating used for flow cytometry sorting of BG stem cells (ITGA6 high /CD34 + ) in telogen (P49) wild-type and NSUN2/ mice. (D) Gating used for flow cytometry sorting of HG progenitor cells (ITGA6 low /PCAD + ) in telogen (P49) wild-type and NSun2/ mice. (E,F) Log2 FC (E) and Gene Ontology categories (F) of combined differential expressed genes (FDR < 0.05) in anagen and progenitor (ITGA6 low /PCAD + ) populations from skin of NSUN2+/+ and / mice. (G) Schematic representation of NSUN-dependent methylation at cytosine-5. (H) Overview of the one-carbon metabolism network. (I-N) Metabolic differences between NSUN2+/+ and NSUN2/ mice relating to the methionine cycle (I,L), free amino acids (J,M), and free nucleotides (K,N) measured by NMR-based (I-K) or MS-based (L-N) metabolic profiling (n = 3–5 mice). (O) Model of how protein homeostasis changes the balance between protein synthesis and degradation in NSUN+/+ (upper panel) and NSUN2/ (lower panel) cells. The underlying data for this figure can be found in S2 Data and S1 File. BG, bulge DP, dermal papilla FC, fold-change FDR, false discovery rate HG, hair germ IFE, interfollicular epidermis ITGA6, integrin alpha-6 MS, mass spectrometry NMR, nuclear magnetic resonance PCAD, P-cadherin SAH, S-adenosyl-homocysteine SAM, S-adenosyl-methionine SG, sebaceous gland.

S2 Fig. Rescue for loss of NSUN2 by reexpressing the wild-type or enzymatic dead protein.

(A, B) Differentially expressed genes in NSUN2−/ compared to NSUN2+/+ cells (A) and Nsun2 RNA levels in NSUN2+/+, +/, and / cells (B) measured by RNA sequencing. (C, D) The transcriptional profile of NSUN2−/ cells overexpressing the NSUN2 protein is largely unaltered (C) although Nsun2 is highly expressed (D). Expression of the empty (‘e.’) vector served as a control. (E) Venn diagram of differentially expressed genes (padj < 0.01) in NSUN2−/ versus +/+ compared to NSUN2-rescued cells. (F) Two out of three replicates of polysome profiles using NSUN2+/+ and / cells. (G) Schematic representation of OP-puro incorporation in actively translating ribosomes. OP-puro mimics an amino-acyl-loaded tRNA molecule. (H) Example raw data outputs from OP-puro fluorescence analysis using a flow cytometer. CHX served as a control. (I) Protein synthesis measured by OP-puro incorporation in NSUN2+/+ and / cells after incubation with an angiogenin inhibitor (ANGi). (J) Western blot for NSUN2 and tubulin after incubation with 500 or 1,000 nm RAPA for 12 or 24 hours (h). (K) Quantification of protein expression shown in (J). (L) De novo protein synthesis in NSUN2+/+ and / after incubation with RAPA or CHX. DMSO served as a vehicle control (J-L). (M, N) Metabolic differences of NSUN2−/ cells rescued with the empty vector (‘e.v.’), K190M, or the NSUN2 protein shown as a PCA plot (M) or as Log2 FC differences of the significant different (p < 0.01 NSUN2 versus e.v.) metabolites (N). The underlying data for this figure can be found in S4 and S7 Data and S1 File. CHX, cycloheximide OP-puro, O-propargyl-puromycin PCA, principle component analysis RAPA, rapamycin tRNA, transfer RNA.

S3 Fig. NSUN2 regulates cell cycle phases and global protein synthesis during the cellular stress response.

(A) Example raw data outputs from OP-puro fluorescence analysis using a flow cytometer for human dermal fibroblasts treated with sodium arsenite. Dotted line represents the mean level of OP-puro positive control. (B) Immunofluorescence detection of OP-puro incorporation in human dermal fibroblasts. DAPI: nuclear counterstain. Scale bar: 20 μm. (C) Measurement of OP-puro fluorescence intensity in cells using microscope-acquired images. Each dot represents one cell. Data are represented as median. (D) Second replicate of polysome profiling of NSUN2+/+ and NSUN2−/ cells rescued with wt or mutated NSUN2 (K190M). The empty vector (‘e.V.’)-infected cells served as control (see Fig 3F–3I). (E) Example of raw data output from AnV and PI analysis to measure cell death. (F, G) Percentage of cells that are viable, apoptotic, or necrotic in NSUN2+/+ and NSUN2−/ cells exposed to sodium arsenite for the indicated hours (hr) (n = 3 samples per time point). (H) Summary of cell cycle distribution shown in Fig 3A–3D. Data represented as mean in (K-H). Error bars are ±SD. The underlying data for this figure can be found in S1 File. AnV, AnnexinV OP-puro, O-propargyl-puromycin PI, propidium iodide wt, wild-type.

S4 Fig. RNA methylation levels change dynamically in response to oxidative stress.

(A) Immunofluorescence detection of the stress granules markers eIF4A1 (upper panels) and p-eIF2A (lower panels) in untreated (control) or sodium arsenite–treated NSUN2+/+ and NSUN2−/ cells. DAPI: nuclear counterstain. Scale, 20 μm. (B) Nsun2 RNA levels in response to UVB exposure in primary human keratinocytes and dermal fibroblasts. (C) Western blot for NSUN2 in NSUN2+/+ and / cells incubated with vehicle control (DMSO, PBS). (D) Experimental outline of sample collection and RNA BS sequencing. (E,F) Quantification of tRNA methylation percentage of NSUN2-dependent (E) and -independent (F) sites in a second independent experiment (n = 5 samples per time point). (G) Second independent RNA BS-seq data shown as heatmap of methylation status of individual tRNA molecules in NSUN2+/+ and NSUN2−/ cells. (H, I) Quantification of methylation changes in the tRNAs Leu CAA and Asp GTC in NSUN2+/+ and NSUN2−/ cells shown in (E). Data represented as median in (E, F, H, I). One-way ANOVA adjusted p-value (H,I), **p < 0.005, ***p < 0.0005, ****p < 0.0001. The underlying data for this figure can be found in S8 Data and S1 File. BS, bisulfite BS-seq, BS sequencing eIF2, eukaryotic Initiation Factor 2 tRNA, transfer RNA.

S5 Fig. Dynamic levels of NSUN2, tRFs, and methylation in response to stress.

(A) Reexpression of NSUN2 but not K190M in NSUN2−/ cells restores methylation to similar levels of endogenous NSUN2 (NSUN2+/+). (B) Western blot for NSUN2 in NSUN2−/ cells infected with an empty (‘E.’) vector control, the enzymatic dead K190M, or the wild-type NSUN2 construct in cells untreated (0) or treated for 2 and 4 hours (h) with sodium arsenite. HSP90 served as a loading control. (C) Raw data (reads) for the indicated tRNAs obtained from small RNA sequencing in NSUN2-expressing (+/+ black) or -lacking (/ red) untreated (0h) or (h) treated 2 and 4 hours with arsenite. (D, E) Methylation levels (pooled from 5 replicates) of cytosines along tRNA 71-Leu CAG (C) and 1-His GTG (F) detecting m 5 C sites, in the variable loop (D) and C70 (E). The underlying data for this figure can be found in S10 Data and S1 File. HSP90, heat shock protein 90 m 5 C, 5-methylcytosine tRF, tRNA-derived fragment tRNA, transfer RNA.

S6 Fig. Site-specific methylation shapes tRF formation to regulate protein synthesis.

(A-C) Comparison of site-specific tRNA methylation and fragmentation in NSUN2+/+ (red) and NSUN2−/ (blue) cells. (n = 4 samples per time point). Data represent median and range. Adjusted p-value: one-way ANOVA. **p < 0.005, ***p = 0.0005. (D) Log2 FC of the down-regulated tRFs when NSUN2-overexpressing cells are exposed to stress for 4 hours. tRNA lysine-derived tRFs are highlighted in red tRNA histidine-derived tRFs are highlighted in blue. Line indicates the mean. (E) Treatment regime to measure global protein synthesis of NSUN2+/+ and / cells transfected with tRNA Glu CTC -derived 5′ and 3′ tRFs after exposure to sodium arsenite. (F, G) Log2 FC of protein synthesis in NSUN2+/+ (F) and NSUN2−/ (G) in response to synthetic 5′ or 3′ tRFs. (n = 3 samples per time point). (H) A fluorescence siRNA was used as a control for transfection efficiency. The underlying data for this figure can be found in S11 and S12 Data and the S1 File. FC, fold-change siRNA, small interfering RNA tRF, tRNA-derived fragment tRNA, transfer RNA.

S7 Fig. Mitochondrial activity is reduced and catabolic pathways enhanced in the absence of NSUN2.

(A, B) Mitochondrial activity (‘mito’) and protein synthesis (‘OP-puro’) after exposure to arsenite for the indicated time (hours) or CHX in NSUN2+/+ (A) and NSUN2−/ (B) cells. (C) GO analyses using Ribo seq data in NSUN2−/ cells rescued with NSUN2 or the enzymatic dead versions of NSUN2 C321A (left panel) and C271A (right panel). (D, E) PCA plot (D) and heatmap (E) of genes belonging to the GO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay. The underlying data for this figure can be found in S15 Data and S1 File. CHX, cycloheximide GO, Gene Ontology OP-puro, O-propargyl-puromycin PCA, principle component analysis seq, sequencing.

S1 Data. Mouse skin microarray complete dataset.

S2 Data. Mass spectrometry raw data.

S2A: Mass spectrometry data for human dermal fibroblasts S2B: Mass spectrometry data for mouse skin S2C: NMR data for mouse skin. NMR, nuclear magnetic resonance.

S3 Data. Multiple t test for metabolites shown in Fig 1I–1K.

S4 Data. RNA sequencing data.

S4A: RNA sequencing data of NSUN2+/+, NSUN2+/, and two lines of NSUN2−/ human dermal fibroblasts. S4B: RNA sequencing data NSUN2−/ cells reexpressing the NSUN2 protein or the empty vector as a control.

S5 Data. RNA bisulfite sequencing of NSUN2−/− cells rescued with the wild-type NSUN2 construct or the enzymatic dead NSUN2 construct K190M, and the empty vector as a control.

S6 Data. Small RNA sequencing of rescued NSUN2−/− cells using the wild-type or enzymatic dead version of NSUN2 or the empty vector as control.

S7 Data. Mass spectrometry data for rescued NSUN2−/− cells using the wild-type or enzymatic dead version of NSUN2 or the empty vector as control.

S8 Data. RNA BS sequencing data.

S8A: BS-seq tRNA methylated sites (n = 4 conversion assays) from first experimental replicate. S8B: BS-seq tRNA methylated sites (n = 5 conversion assays) from second independent experimental replicate. BS, bisulfite BS-seq, BS sequencing tRNA, transfer RNA.

S9 Data. RNA BS sequencing data identifying other potentially non–tRNA targeted sites by NSUN2.

S9A: BS-seq to detect other potentially non–tRNA targeted sites by NSUN2 from 1 replicate (n = 4 conversion assays). S9B: BS-seq to detect other all sites not targeted by NSUN2 from 1 replicate (n = 4 conversion assays). BS, bisulfite tRNA, transfer RNA.

S10 Data. RNA BS sequencing data of unstressed and stressed NSUN2−/− cells infected with the empty vector control, the wild-type NSUN2, or the enzymatic dead construct K190M.

S11 Data. Small RNA sequencing in unstressed and stressed cells.

S11A: tRNA fragments found in the small RNA-seq dataset. S11B: tRNA fragment sequencing and analysis (p < 0.01 NSUN2−/ at 2 hours of stress). S11C: tRNA fragment sequencing and analysis (samples with missing values removed). S11D: tRNA fragment sequencing and analysis (fragments smaller than 40 nucleotides). RNA-seq, RNA sequencing tRNA, transfer RNA.

S12 Data. tRNA fragments in NSUN2, K190M, or empty vector infected cells after 0, 2, and 4 hours of treatment with sodium arsenite.

S13 Data. RNA sequencing data.

S13A: RNA-seq data from human dermal fibroblasts untreated (‘ctr’) or treated with sodium arsenite for 2 or 4 hours. Shown are transcriptional differences between NSUN2−/ and NSUN2 +/+ cells in the control. S13B: RNA-seq data from human dermal fibroblasts untreated (‘ctr’) or treated with sodium arsenite for 2 or 4 hours. Shown are transcriptional differences between NSUN2−/ and NSUN2 +/+ cells after 2 hours of stress. S13C: RNA-seq data from human dermal fibroblasts untreated (‘ctr’) or treated with sodium arsenite for 2 or 4 hours. Shown are transcriptional differences between NSUN2/ and NSUN2 +/+ cells after 4 hours of stress. RNA-seq, RNA sequencing.

S14 Data. Gene ontology analyses.

S14A: Gene enrichment (Cellular processes_Gorilla) for differentially expressed genes in NSUN2+/+ versus NSUN2−/ cells after 2 hours of exposure to sodium arsenite. S14B: Gene enrichment (Cellular processes_Gorilla) for differentially expressed genes in NSUN2+/+ versus NSUN2−/ cells after 4 hours of exposure to sodium arsenite. S14C: Gene enrichment (Cellular processes_Gorilla) for differentially expressed genes in NSUN2+/+ versus NSUN2−/ cells in untreated condition revealed no significant enrichment.