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Sample GSM3211650 Query DataSets for GSM3211650
Status Public on Jun 25, 2018
Title Amplicon_BS_2i_430
Sample type SRA
Source name E14 ESC
Organism Mus musculus
Characteristics cell type: Embryonic stem cell
cell line: E14 ESC
strain: 129/Ola
Growth protocol Naïve ESCs were cultured in serum free media with 2i inhibitors (Neurobasal, N2, B27, 103 U/ml LIF, 1 μM Mek inhibitor PD0325901 and 3 μM Gsk3β inhibitor CHIR99021) without feeders at 37°C and 5% CO2. Primed ESCs were cultured in serum containing media (DMEM 4,500 mg/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 15% fetal bovine serum, 1 U/ml penicillin, 1 μg/ml streptomycin, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, and 103 U/ml LIF ESGRO) without feeders at 37°C and 5% CO2.
Extracted molecule genomic DNA
Extraction protocol Cells were lysed by removing media from culture dishes and adding 200ul of RLT plus buffer (Qiagen) supplemented with 0.5mM 2-mercaptoethanol. . Total nucleic acid was purified from cell lysates with RNAdvance magnetic beads (Beckman Coulter, A32649) using a Bravo Workstation pipetting robot (Agilent Technologies) following the manufacturers protocol. RNA was subsequently purified by treating the total nucleic acid with DNase I.
KAPA HiFi Uracil+ Master Mix (Kapa Biosystems) was used to amplify regions of interest using 30nM primers (Table S3), with the reverse primer including an 8N unique molecular identifier (UMI). The PCR program was: 95C 5min; 35 repeats of 98C 20s, 60C 15s, 72C 60s; 72C 10min. Amplicons were pooled for each sample and purified using Ampure XP beads (Agencourt), before a second round of PCR was used to incorporate Illumina Adaptor sequences and index samples. The PCR reaction included 11ul pooled amplicons, 200nM indexed PE1.0 (Table S5) and iPCRTag primers (Quail et al., 2012), and KAPA HiFi Master Mix (Kapa Biosystems). The PCR program was: 98C 45s; 5 repeats of 98C 15s, 65C 30s, 72C 30s; 72C 5min. Samples were then pooled and purified before library QC and sequencing was performed with up to 144 samples included on a 150bp paired-end MiSeq run.
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina MiSeq
Description library strategy: Bisulfite-Seq amplicon-Seq
Data processing Libraries were sequenced on the Illumina HiSeq platform using the default RTA analysis software.
The first 8 bp of Read 2, representing unique molecular identifiers (UMIs), were removed and written into the sequence IDs reads instead. The reads were then subjected to adapter and quality trimming using Trim Galore (v0.4.2; default parameters) and aligned to the mouse genome (build GRCm38) using Bismark (v0.16.3, parameters: --non_directional). After mapping reads were deduplicated using the UMI-mode of deduplicate_bismark (options --bam --barcode).
Genome_build: GRCm38
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 Jun 22, 2018
Last update date Jun 26, 2018
Contact name Felix Krueger
Organization name Altos Labs
Department Bioinformatics
Street address Granta Park
City Cambridge
ZIP/Postal code CB21 6GP
Country United Kingdom
Platform ID GPL16417
Series (2)
GSE75975 Genome-scale DNA methylation oscillations in pluripotent cells
GSE116163 DNA methylation oscillations at CpG poor enhancers in primed embryonic stem cells [amplicon-Seq]
BioSample SAMN09470973
SRA SRX4284141

Supplementary file Size Download File type/resource
GSM3211650_Amplicon_BS_2i_430.cov.gz 8.1 Kb (ftp)(http) COV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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