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| Status |
Public on Mar 15, 2024 |
| Title |
Nuclei, mock-treated, rep2 |
| Sample type |
SRA |
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| Source name |
W303 background
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
tissue: W303 background
cell line: strain YDC111
cell type: haploid
genotype: MATa ade2-1 can1-100 HIS3 leu2-3,112 trp1-1 ura3-1
treatment: Mock-treated
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| Treatment protocol |
Spheroplasts were prepared from ~100 A600 units of cells in 15 ml SM Buffer (SC medium with 1 M sorbitol, 50 mM Tris-HCl pH 8.0, 20 mM 2-mercaptoethanol) by digestion with ~19,000 units of lyticase (Sigma L-2524) at 30°C for 10 min. Digestion of the cell wall was monitored by measuring the A600 of 30 μl cell suspension in 1 ml 1% SDS and considered complete when the A600 decreased to < 10% of the initial value. Spheroplasts were spun down in a pre-cooled Sorvall SA600 rotor (7,500 rpm for 5 min at 4°C) and washed once with 25 ml cold ST Buffer (1 M sorbitol, 50 mM Tris-HCl pH 8.0). Spheroplasts were lysed by resuspension in 20 ml cold F Buffer (18% w/v Ficoll-PM400 (GE Healthcare 17-0300-50), 40 mM potassium phosphate, 1 mM magnesium chloride, pH 6.5; protease inhibitors (Roche 05056489001) and 5 mM 2-mercaptoethanol were added just before use). The lysate was applied to a step gradient of 15 ml cold FG Buffer (7% w/v Ficoll-PM400, 20% glycerol, 40 mM potassium phosphate, 1 mM magnesium chloride, pH 6.5, with protease inhibitors and 5 mM 2-mercaptoethanol as above) and spun in the SA600 rotor (12,500 rpm for 20 min at 4°C). To perform methylation, the pellet of crude nuclei was resuspended in 4.8 ml pre-warmed NEB buffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 0.1 mg/ml BSA, pH 7.9) with 1.2 mM S-adenosylmethionine (SAM) and protease inhibitors, and divided into ten 425 µl aliquots. M.EcoGII (24 µl; 600 units at 25 units/µl; New England Biolabs M0603B-HC1) or 24 µl water was added to each of five aliquots, mixed thoroughly but gently, and incubated at 30°C for 20 min. Methylation was stopped by adding 50 μl 200 mM EDTA, 10% SDS and incubating at 25°C for 5 min. Genomic DNA was purified by addition of 27.5 μl 10% SDS, mixing, addition of 133 μl 5 M potassium acetate, followed by two extractions with an equal volume of chloroform, precipitation with 0.7 vol. isopropanol, and one wash with 75% ethanol. The purified genomic DNA was dissolved in 20 μl 10 mM Tris-HCl pH 8.0, 0.5 mg/ml RNase A and incubated at 37°C for 4 h. To verify methylation, purified genomic DNA was digested with either DpnI, which only cleaves when its recognition site (GATC) is methylated, or MboI, which recognises the same site but is blocked by methylation, and evaluated by gel electrophoresis. Genomic DNA positive control samples (gDNA) were obtained by subjecting genomic DNA purified from yeast cells as above to three rounds of M.EcoGII treatment to maximise methylation (28 μg DNA in 160 μl NEB buffer 2.1 with 0.8 mM SAM, incubated with 96 units M.EcoGII at 37oC for 4 h). The DNA was purified after each round.
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| Growth protocol |
Yeast strain YDC111 was grown at 30°C in synthetic complete (SC) medium (2% D-glucose, 6.7 g/l yeast nitrogen base (Sunrise Science Products 1501-250) and 0.79 g/l Complete Supplement Mixture with adenine (Sunrise Science Products 1128-010) to an OD A600 ~0.2 and arrested in G1 by the addition of ɑ-factor (FDA Core Facility) to 10 μg/ml. Arrest was monitored by observance of the "shmoo" phenotype in a light microscope. After 2.5 h, the cells were harvested by filtration and stored at -80°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA samples were sheared to ~2 kb in a total volume of 200 µl using a Covaris miniTube Clear on the Covaris M220 with shearing conditions as follows: Peak Incident Power: 8 W, Duty Factor: 20%, Cycles per Burst: 1000, Treatment time: 900 s. Sonicated DNA was purified using AMPure XP beads (Beckman-Coulter) at a 1:1 ratio, eluted in 22.5 µl AMPure Elution Buffer and checked for quality using the DNA12000 assay in an Agilent BioAnalyzer.
SMRTbell libraries were prepared using PacBio barcoded adapters according to the manufacturer's instructions.
PacBio long-read SMRT sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Sequel |
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| Data processing |
The raw pacbio subreads are called using the default pipeline by BLASR using SMRT analysis 6.0 with default settings, and the reads are aligned using pbalign using the SMRT Analysis 10.2 software.
The aligned subreads were grouped by ZMW and we predicted consensus m6A sites for each ZMW using the ipdSummary package from SMRT Analysis 10.2 with default parameters. We also computed the CCS reads from the raw subreads using the SMRT Analysis 10.2 with default parameters, and filtered the ZMWs with minimal average base quality score=90 in the CCS reads.
Supplementary files format and content: Bam format (with _m6A suffix). The m6A sites in the consensus reads are annotated as insertions at A sites, and the subread coverage of the consensus read is stored in the dp tag.
We predicted nucleosome footprints and accessible regions in each read using our own program (described in the paper), and stored the per-read nucleosome footprints and accessible regions in bed files and bam files.
Note from submitter: The . bam files allow the user to visualise our nucleosome predictions in a viewer such as IGV. These processed bam files are much more informative than the bed files: m6A bases are annotated as insertions; nucleosomes are annotated as substitutions; ambiguous regions are annotated as deletions, and accessible regions are annotated as matches. These files show both the data and the predictions of our bioinformatic pipeline and represent the main point of our paper.
Assembly: sacCer3 for yeast data and SmaI linearized pUC19 for pUC19 data
Supplementary files format and content: Bed format. Each track represents one consensus read; accessble regions and nucleosomes are annotated as blocks in each track.
Supplementary files format and content: Bam format (with _Nucleosome suffix). More detailed information is stored In the bam file: m6A sites are annotated as insertions; nucleosomes are annotated as substitutions; ambiguous regions are annotated as deletions, and accessible regions are annotated as matches, suitable for IGV (2.9.2) visualization.
Supplementary files format and content: Bigwig format. The average m6A fraction at in reference location.
Library strategy: m6A-Seq
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| Submission date |
Sep 13, 2023 |
| Last update date |
Oct 15, 2024 |
| Contact name |
David J. Clark |
| E-mail(s) |
clarkda@mail.nih.gov
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| Organization name |
NIH
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| Department |
NICHD
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| Lab |
Clark Lab
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| Street address |
6 Center Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL32099 |
| Series (1) |
| GSE243114 |
A bioinformatic pipeline for analysis of M.EcoGII methylation footprint PacBio long-read sequence data |
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| Relations |
| BioSample |
SAMN37386992 |
| SRA |
SRX21773480 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7779507_Nuclei_NoMEcoGII_Rep2_m6A.bam |
6.0 Mb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
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