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| Status |
Public on May 19, 2017 |
| Title |
D1L |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
background: J1
genotype: Dnmt2-/-
fraction: long RNA fraction (>200 nt) Ribo-
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| Treatment protocol |
The feeder cells were removed by serial passaging as follows: MEFs and ESCs were harvested and the feeder was allowed to attach to the dishes for 40- 60 min at 37 °C. All cells that had not attached were re-plated into second dishes for an additional 40-60 min. The supernatants (non-attached cells) were then spun down and pellets were snap-frozen in liquid nitrogen. This allowed harvest of pure ESCs without any MEF contaminants, as MEFs attach significantly faster. The purity of the harvest was very high, which was ultimately confirmed by genotyping of Dnmt2-/- ESCs using PCR.
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| Growth protocol |
ESCs were grown on a primary MEF feeder layer in standard culture medium: knock-out DMEM (Gibco), 15% heat inactivated fetal calf serum (Hyclone), 1% non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco), 2nM L-glutamine (Gibco), 0.1mM _-mercaptoethanol, 500 U/mL ESGRO (ESG1106, Merck Millipore).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed according to the Invitrogen TRIzol RNA isolation protocol (Ambion). The nucleic acid concentrations and purity were analyzed on the NanoDrop ND-1000 Spectrophotometer. RNA integrity was measured on a Bioanalyzer (Agilent) or TapeStation (Agilent). Samples were stored at -80 °C.
30 µg of total RNA was fractionated into a long (<200 nt) and a small RNA fraction (<200 nt) by a modified protocol for small RNA cloning (W. Gu and D. Conte) using mirVana Isolation Kit buffers. Briefly, 400 μl of Lysis/Binding Buffer was mixed with 48μl of Homogenate Additive and 80 µl of total RNA. The mix was incubated at RT for exactly 5 min. 1/3rd volume of Ethanol was added and mixed by inversion. This was incubated at RT for exactly 20 min to increase yield. 10 μg GlycoBlue was added and spun at 21 °C and 2,500 x g for 8 min to pellet the long fraction. The supernatant containing the small fraction was carefully transferred to a fresh tube and precipitated with isopropanol and 15 μg GlycoBlue at -80 °C for at least 10 min. rRNA depletion was carried out on the small fraction and on the half amount of the long fractions according to the RiboMinus Eukaryote System v2 (Ambion) protocol. The other half amount of the long fraction was further processed and sequenced as Ribo+ sample. rRNA-depleted samples were concentrated using ethanol precipiptation. The long fractions were further processed with the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs). This was carried out as described in the manual. 3 min of fragmentation at 94 °C has been established to lead to a peak at approximately 250 nt, appropriate for the final 100 bp paired-end sequencing. The fragmented RNA was precipitated using ethanol with 20 μg GlycoBlue at -80 °C for at least 10 min. TURBO DNase (Ambion) was applied to each sample according to the protocol in a total volume of 20 μl. This volume was immediately applied to the EZ RNA Methylation Kit (Zymo Research), following the manufacturer’s manual. As a final step before library preparation, a stepwise RNA end repair was carried out using T4 polynucleotide kinase (TaKaRa). 3’-Dephosphorylation and 5’-phosphorylation (using 10 mM dATP) were carried out at 37°C for 20 min each. RNA was further purified by a phenol/chloroform extraction and ethanol precipitation. Library preparation was done according to the NEBNext Small RNA Library Prep Set protocol. Following adaptor ligation and cDNA synthesis, cDNA was amplified with 12 cycles of PCR and purified using the QIAquick PCR Purification Kit (Qiagen). The libraries were further size selected on a 6% polyacrylamide gel. Compatible barcodes were selected and samples were pooled in equimolar ratios on multiple lanes on an Illumina HiSeq 2000 platform. A 100 bp paired-end sequencing approach was used.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Library strategy: RNA Bisulfite-seq
Reads were trimmed with a quality cutoff of 30 for each base (corresponding to a confidence level of 99.9%) and aligned using BSMAP (Xi and Li 2009), which allows C-T base modifications without flagging them as mismatches.
We used 3% mismatch rate, and full sequence usage in BSMAP (parameters: -s 12 -v 0.03 -g 0 -w 1000 -S 0 -p 1 -V 1 -I 1 -n 0 -r 2 -u -m 15 -x 1000).
Resulting aligned reads were kept only if their length was 25 nt or more, if they aligned uniquely, and if both forward and reverse pairs were located at the same positions.
Reads containing more than 3 adjacent non-converted cytosines (rRNA and mRNA), or more than 6 adjacent non-converted cytosines (tRNA) were interpreted as non-converted reads resulting from RNA folding or bound proteins and were thus discarded. The presence of Nsun2- and Dnmt2-dependent methylation at C38, C48 and C49 in some tRNAs made it necessary to raise this threshold from 3 to 6 in tRNAs. (2.) Mismatches apart from C/T, occurring consistently at a specific position indicate the existence of a distinct subset of reads (as caused by RNA editing or single-nucleotide polymorphisms). If such a subset additionally correlates with the presence or absence of a converted cytosine, we discarded the mismatched subsets of reads to avoid confounding errors (upon significance of a Fisher exact test between reads with converted cytosines or not, compared to reads with a mismatch or not, Bonferroni-adjustment for multiple testing, significance cutoff α=0.05). We permitted an exception for tRNA(Val), which is known to be subject to RNA editing at position 34
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include non-conversion ratio at each cytosine
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| Submission date |
May 24, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesca Tuorto |
| E-mail(s) |
[email protected]
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| Organization name |
Deutsches Krebsforschungszentrum
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| Department |
Division of Biochemistry Mannheim Institute for Innate Immunoscience (MI3)
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| Street address |
Ludolf-Krehl-Str. 13-17
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| City |
Mannheim |
| ZIP/Postal code |
D-68167 |
| Country |
Germany |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE81823 |
Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs [Whole-transcriptome bisulfite sequencing] |
| GSE81825 |
Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs |
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| Relations |
| BioSample |
SAMN05171584 |
| SRA |
SRX1797735 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2176376_D1L.txt.gz |
15.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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