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Sample GSM2299703

Query DataSets for GSM2299703

Status

Public on May 15, 2017

Title

RNA_40_45A

Sample type

SRA

Source name

40-45um oocytes

Organism

Mus musculus

Characteristics

background strain: C57BL/6
developmental stage: Oocytes
genotype: Wild-type

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using TRIsure reagent (Bioline) followed by RNA Clean & Concentrator (Zymo Research) with on-column DNAse treatment (RNasefree DNase I, Life Technologies). Reverse transcription was performed using SuperScript III (Life Technologies), followed by second DNA strand synthesis using dUTPs instead of dTTPs and DNA polymerase I (NEB); libraries were constructed using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB), including dUTP excision step by USER Enzyme (NEB) before PCR.
RNA was extracted using TRIsure reagent (Bioline) followed by RNA Clean & Concentrator (Zymo Research) with on-column DNAse treatment (RNasefree DNase I, Life Technologies).
For RNA-seq libraries: Reverse transcription was performed using SuperScript III (Life Technologies), followed by second DNA strand synthesis using dUTPs instead of dTTPs and DNA polymerase I (NEB); libraries were constructed using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB), including dUTP excision step by USER Enzyme (NEB) before PCR. RRBS: Extracted DNA was spiked with a small amount of lambda DNA (0.05pg per 6ng of genomic DNA) for bisulphite conversion control, digested with MspI endonuclease (Fermentas), end-repaired (Klenow fragment exo-, Fermentas, with 10nM dATP, 1nM dCTP and 1nM dGTP) and ligated with 5mC-adaptors (Illumina) with T4 ligase (Fermentas). Bisulphite conversion was done using the EZ DNA Methylation-Direct Kit (Zymo Research) and DNA was amplified by 18 cycles of PCR using Pfu Turbo Cx polymerase (Stratagene). DNA was purified using SPRI beads (Agencourt). For PBAT: As in Stewart et al. (2015): Cells were lysed in EB buffer (Qiagen) with 0.5% SDS (Sigma, 161-0418) and bisulfite-treated using theone-stepmodification procedure in the Imprint DNA modification kit(Sigma,MOD50-1KT).The resulting DNA was purified using the EZ DNA methylation direct kit (Zymo, D5020). First strand synthesis was performed using Klenow Exo− (New England Biolabs, M0212S) and a custom streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs (bp) of random sequence (9N). This was followed by exonuclease I treatment (New England Biolabs, M0293S), purification using SPRI beads (1.8× ratio; Fisher Scientific, 09-981123), and binding to biotin beads (Life Technologies, 11205D). Samples were then subjected to second strand synthesis, again using custom primer, followed by 10 cycles of library amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1000

Data processing

RRBS reads were trimmed to remove poor quality calls and adapters using Trim Galore v0.3.5 (parameters --rrbs) and mapped to the mouse genome GRCm38 assembly by Bismark (Krueger and Andrews, 2011) v0.14.0 (options --phred64-quals). For PBAT data, trimmed reads (Trim Galore v0.3.5 using default parameters) were first aligned to GRCm38 in paired-end mode to be able to count overlapping parts of the reads only once while writing out unmapped singleton reads; in a second step remaining singleton reads were aligned in single-end mode. Alignments were carried out with Bismark v0.10.0 with the following set of parameters: --pbat for paired-end mode, --pbat for single-end mode for read 1 and default parameters for single-end mode read 2. Reads were then deduplicated with Bismark selecting a random alignment for positions that were covered more than once. CpG, CHH and CHG methylation calls were extracted using the Bismark methylation extractor (v0.10.0) with the following parameters: --no_overlap --report --ignore 4 --ignore_r2 4 for paired-end mode and --report --ignore 4 for the single-end mode. Published bisulphite-sequencing data were processed as described previously (Veselovska). Raw RNA-seq reads were trimmed to remove both poor quality calls and adapters using TrimGalore v.0.2.8 and mapped to GRCm38 using TopHat v.2.0.9 (option –g 1).
Genome_build: GRCm38
Supplementary_files_format_and_content: Read counts (RPKM-like) RNA-Seq data file is comma delimited and contain the following columns: (1) Gene ID,(2) Chrromosome,(3) Start,(4) End,(5) Strand,(6-14) Sample Read Counts. The values were corrected for the length
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>. It combined both the single-end and paired-end alignments.

Submission date

Aug 31, 2016

Last update date

May 15, 2019

Contact name

Gavin Kelsey

Organization name

The Babraham Institute

Street address

Babraham Research Campus

City

Cambridge