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Sample GSM4367039 Query DataSets for GSM4367039
Status Public on Jun 22, 2020
Title primed_H9KN2_replicate1
Sample type SRA
Source name H9-KN2 human embryonic stem cell line
Organism Homo sapiens
Characteristics cell type: embryonic stem cells
cell line: H9-KN2 human embryonic stem cell line
protocol: cultured in primed conditions
Treatment protocol 1μg of genomic DNA was bisulfite converted using the EZ DNA methylation Gold kit (Zymo Research) according to manufacturer’s instructions with either magnetic bead or column clean-up and eluted/resuspended in 66μl elution buffer to obtain a final concentration of ~15ng/μl bisulfite converted DNA.
Extracted molecule genomic DNA
Extraction protocol [Sample collection protocol] Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) or the AllPrep DNA/RNA Micro Kit (Qiagen) according to manufacturer’s instructions and eluted into TE buffer or H2O.
The IMPLICON protocol consists of 2 PCR reactions. In the first reaction each sample is amplified with each primer pair in individual reactions: 30ng (2μl of 66μl eluted bisulfite treated DNA) of bisulfite treated DNA is amplified with 1.2μl of 10μM primer pool (final 1.5μM) containing both forward and reverse primers in 4μl of 2x KAPA HiFi Uracil+ ReadyMix in a final volume of 8μl. DNA was amplified using the following conditions: initial denaturation at 95°C for 5min, 30 cycles of 98°C denaturation for 20 seconds, variable annealing temperature for 15 seconds and extension at 72°C for 60 seconds; followed by a final extension at 72°C for 10 minutes. All PCR reactions for each individual sample were pooled together and samples cleaned-up using 1.5x AMPure XP beads and eluted in 20μl H2O. In the second PCR reaction, barcoded Illumina adapters are attached to the pooled PCR samples ensuring that each sample pool receives a unique reverse barcoded adapter. The 20μl PCR pool was amplified using 1μl of 10μM Illlumina PE1.0 primer (same for all samples) and 1μl of 10μM Illumina iTAG primer (distinct for each sample) and 25μl 2x KAPA HiFi Uracil+ ReadyMix in a 50μl volume reaction using the following conditions: initial denaturation at 98°C for 45 seconds, 5 cycles of 98°C denaturation for 15 seconds, 65°C annealing for 30 seconds and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 5 minutes. Reactions were cleaned-up with 1x AMPure XP beads and eluted in 20μl. Libraries were verified by running 1:30 dilutions on Agilent bioanalyzer.
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina MiSeq
Data processing Standard Illumina base-calling pipeline.
As first step in the processing, the first 8 bp of Read 2 were removed and written into the readID of both reads as an in-line barcode, or UMI (Unique Molecular Identifier). This UMI was then later used during the de-duplication step with "deduplicate bismark --barcode mapped_file.bam".
Raw sequence reads were then trimmed to remove both poor quality calls and adapters using Trim Galore (v0.6.2 for hybrid mouse tissues, v0.4.4 for human and inbred mouse tissues,, Cutadapt version 2.3 for hybrid mouse tissues, v1.9.1 for human and inbred mouse tissues), parameters: --paired). Trimmed reads were aligned to the mouse or human reference genome in paired-end mode. Alignments were carried out with Bismark v0.14.4 for hybrid mouse tissues and v0.18.2 for human and inbred mouse tissues (Krueger and Andrews, 2011) CpG methylation calls were extracted from the mapping output using the Bismark methylation extractor (v0.22.1 for hybrid mouse tissues, v0.18.2 for human and inbred mouse tissues). Deduplication was then carried out with deduplicate_bismark, using the --bacode option to take UMIs into account (see above)). For hybrid mouse strain experiments, the data was aligned to a hybrid genome of Black6/CAST (the genome was prepared with the SNPsplit package (v0.3.4, Following alignment and deduplication, reads were split allele-specifically with SNPsplit.
Genome_build: GRCm38
Genome_build: GRCh38
Supplementary_files_format_and_content: [Bismark coverage file] 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 Feb 28, 2020
Last update date Jun 22, 2020
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 GPL15520
Series (1)
GSE146129 IMPLICON: a high-resolution method to uncover DNA methylation at imprinted regions
BioSample SAMN14247828
SRA SRX7820837

Supplementary file Size Download File type/resource
GSM4367039_primed_H9KN2_replicate1.cov.gz 26.4 Kb (ftp)(http) COV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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