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Status |
Public on Jun 25, 2018 |
Title |
Amplicon_BS_2i_220 |
Sample type |
SRA |
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Source name |
E14 ESC
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Organism |
Mus musculus |
Characteristics |
cell type: Embryonic stem cell cell line: E14 ESC strain: 129/Ola
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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.
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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.
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Library strategy |
Bisulfite-Seq |
Library source |
genomic |
Library selection |
RANDOM |
Instrument model |
Illumina MiSeq |
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Description |
library strategy: Bisulfite-Seq amplicon-Seq
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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>
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Submission date |
Jun 22, 2018 |
Last update date |
Jun 26, 2018 |
Contact name |
Felix Krueger |
E-mail(s) |
fkrueger@altoslabs.com
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Organization name |
Altos Labs
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Department |
Bioinformatics
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Street address |
Granta Park
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City |
Cambridge |
ZIP/Postal code |
CB21 6GP |
Country |
United Kingdom |
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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] |
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Relations |
BioSample |
SAMN09471050 |
SRA |
SRX4284118 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3211627_Amplicon_BS_2i_220.cov.gz |
6.3 Kb |
(ftp)(http) |
COV |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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