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Sample GSM5776324

Query DataSets for GSM5776324

Status

Public on Mar 22, 2023

Title

CFG2353 PM18_D28_5F-K_PEF_RRBS

Sample type

SRA

Source name

PBMC reprogrammed iMSCs

Organism

Homo sapiens

Characteristics

reprogram factors: 4FnoK
reprogram day: day 28

Growth protocol

After reprogramming, the cells were seeded onto fibronectin-coated plates in the mixture of erythroid medium and MSC medium (50:50 ratio) supplemented with small molecules including 3 μM CHIR99021, 10 μM Forskolin, 10 μM ALK inhibitor (SB431542), and 5 μM Tranylcypromine hydrochloride for two days, and replaced with MSC medium thereafter.

Extracted molecule

genomic DNA

Extraction protocol

QIAGEN AllPrep DNA/RNA/miRNA Universal Kit protocol
RNA-seq libraries was constructed using the NuGEN Ovation universal RNA-seq kit (TECAN). RRBS libraries were constructed following standard protocols of the NuGEN Ovation Ultralow Methyl-seq Library Systems. ATAC-seq libraries were constructed by following the improved protocol as described by Corces et al., 2017.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

Reduced Representation

Instrument model

Illumina HiSeq 4000

Description

PM18_D28_5F-K_PEF_RRBS

Data processing

Basecalling, fastq files were generated using Illumina bcl2fastq package v1.8.4 with default settings.
For RNA-seq, the raw reads were trimmed using TrimGalore (v.0.4.5), and then aligned to the human reference genome (GRCh38) for gene counts using STAR (--quantMode GeneCounts).
For ATAC-seq, the raw reads were mapped to human reference genome (GRCh38) using the bioinformatics pipeline snakePipes with the ATAC-seq mode. In the pipeline, fastq files were trimmed using TrimGalore, aligned with Bowtie2, and peaks are called with MACS2.
For ATAC-seq, a consensus peak set was defined by taking the intersection of peaks from both biological replicates using soGGi (v.1.18) package and regions intersecting with blacklisted regions and ChrY were excluded. The ATAC-seq count matrix was computed by counting the reads that fell into the consensus peaks using Rsubread v2.0.1.
For RRBS analysis, raw fastq reads were trimmed using TrimGalore (v.0.4.5) and Nugen RRBS trimming script (trimRRBSdiversityAdaptCustomers.py). After trimming, reads were aligned to the human reference genome (GRCh38) with Bismark54 by default parameter settings, and PCR duplicates were removed by Nugen script (nudup.py).
The methylation call files (Bismark files) including the location of each CpG sites and the methylation percentage were generated by the bismark_methylation_extractor function.
Genome_build: NCBI GRCh38
Supplementary_files_format_and_content: For ATAC-seq datasets, narrowpeak files were generated. For RRBS datasets, CpG coverage report in text format were generated.

Submission date

Jan 07, 2022

Last update date

Mar 22, 2023

Contact name

Charles Wang

E-mail(s)

wchen@llu.edu

Organization name

Loma Linda University

Street address

11021 Campus Street