 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 20, 2020 |
| Title |
oGV_26 [BiSulfite-seq] |
| Sample type |
SRA |
| |
|
| Source name |
germinal vesicle (GV) oocyte
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6Babr
developmental stage: germinal vesicle (GV)
age group: old
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Oocytes were released from the ovaries mechanically with a fine needle and cleaned in M2 medium (Sigma). Clean oocytes were washed twice in PBS, inspected to ensure complete removal of adherent granulosa cells, then individually lysed and flash-frozen in 5μl RLT Plus buffer (Qiagen) and stored at -80°C until further use.
DNA and RNA from individual oocytes were physically separated using the G&T protocol (Angermueller et al., 2016). Briefly, Smart-seq2 oligo-dTs (Picelli et al., 2013, 2014) were annealed to magnetic beads (MyOne C1, Life Technologies) and used to capture polyadenylated mRNA from individual oocyte lysates. The remaining lysate containing the DNA was transferred to a separate tube and the beads washed three times in 1xFSS buffer (Superscript II, Invitrogen), 10 mM DTT, 0.005% Tween-20 (Sigma) and 0.4 U μl−1 of RNAsin (Promega). The washing solutions were added to the DNA tube to maximise recovery. mRNA on the beads was immediately processed further for cDNA conversion by resuspending beads in 10 μl of reverse transcriptase mastermix (100 U SuperScript II (Invitrogen), 10 U RNAsin (Promega), 1 × Superscript II First-Strand Buffer, 2.5 mM DTT (Invitrogen), 1 M betaine (Sigma), 9 mM MgCl2 (Invitrogen), 1 μM Template-Switching Oligo (TSO, Exiqon (Picelli et al., 2013, 2014)), 1 mM dNTP mix (Roche)). The mRNA mixture was reverse transcribed by incubation for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C. The cDNA was amplified by PCR by adding 11 μl of 2 × KAPA HiFi HotStart ReadyMix and 1 μl ISPCR primer (2 μM; (Picelli et al., 2013, 2014)). Samples were incubated in a thermocycler at 98 °C for 3 min, followed by 18 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and finally 72 °C for 5 min. The amplified product was purified using Ampure XP beads with a 1:1 ratio and eluted into 20 μl of water. In parallel, the lysate containing the DNA was purified using a 0.8:1 volumetric ratio of Ampure XP Beads (Beckman Coulter) and eluted into 10 μl of water to use for scBS-seq library preparation.
Single-cell BS-seq (scBS-seq) libraries were prepared as previously described (Smallwood et al., 2014), with minor changes. DNA purified from single cells was bisulphite converted using the EZ Methylation Direct kit (Zymo) according to the manufacturer’s instructions, but using half volumes. Five rounds of first-strand synthesis were carried out, starting by eluting bisulphite-converted DNA into 40 μl of first-strand synthesis mastermix (1 × Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4μM 6NF oligo (IDT)). The mixture was heated to 65 °C for 2 min and cooled on ice. 50U of Klenow exo- (Enzymatics) was added and the mixture incubated on a thermocycler at 37 °C for 30 min after slowly ramping from 4 °C. First-strand synthesis was repeated 4 more times with the addition of 2.5 μl of reaction mixture (1× blue buffer, 0.25 mM dNTPs, 10 mM 6NF oligo and 25U Klenow exo‑). In the final round, samples were incubated for 90 min at 37 °C. Exonuclease digestion was carried out by adding 20U of exonuclease I (NEB) to the reactions, diluting with water to a total volume of 100 μl, followed by incubation at 37 °C for 1 hour. Samples were purified with AMPure XP beads using a 0.75:1 ratio. Beads were resuspended in 50 μl second-strand master mix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT). Reactions were heated for 98 °C for 1 min, cooled on ice, before adding 50U of Klenow exo- (Enzymatics). The mixture was incubated on a thermocycler at 37 °C for 90 min after slowly ramping from 4 °C. Samples were purified using a 0.75:1 ratio of AMPure XP beads and libraries were amplified in 50 μl of PCR mastermix (1× KAPA HiFi Readymix, 0.2 μM PE1.0 primer, 0.2 μM iTAG index primer) using the following protocol: 2 min at 95 °C, 14 cycles of 80 sec at 94 °C, 30 sec at 65 °C, 30 sec at 72 °C and final extension for 3 min at 72 °C. Finally, scBS-seq libraries were purified using a 0.7:1 volumetric ratio of AMPure XP beads and eluted in 15 µl EB buffer before pooling and sequencing.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Libraries were sequenced on the Illumina Nextseq500 platform using the default RTA analysis software.
Alignment was performed in single-end non-directional mode followed by deduplication (deduplicate_bismark) and methylation calling (bismark_methylation_extractor) using Bismark (v0.18.2). The methylation calls for both Read 1 and Read 2 were then merged afterwards using a Python script.
Libraries having a mapping efficiency <10%, fewer than 500,000 CpGs covered, or greater than 50% methylated CpGs were discarded
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
|
| |
|
| Submission date |
Jul 12, 2020 |
| Last update date |
Nov 20, 2020 |
| Contact name |
Felix Krueger |
| E-mail(s) |
[email protected]
|
| Organization name |
Altos Labs
|
| Department |
Bioinformatics
|
| Street address |
Granta Park
|
| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE154271 |
Increased transcriptome variation and localised DNA methylation changes in oocytes from aged mice revealed by parallel single-cell analysis [scBS-seq] |
| GSE154370 |
Increased transcriptome variation and localised DNA methylation changes in oocytes from aged mice revealed by parallel single-cell analysis |
|
| Relations |
| BioSample |
SAMN15513941 |
| SRA |
SRX8714183 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4668140_oGV_26.cov.gz |
21.7 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
