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Sample GSM3045220

Query DataSets for GSM3045220

Status

Public on Aug 11, 2020

Title

BOE-1_RNA-seq

Sample type

SRA

Source name

H7 hESC-CM, BANCR overexpression

Organism

Homo sapiens

Characteristics

tissue: H7 hESC-CM
gender: female
differentiation days: Day 20-30

Treatment protocol

Cardiomyocytes were differentiated from human and NHP pluripotent cells. Briefly, differentiation medium consisted of RPMI-1640 media (11875-085, Life Technologies) supplemented with B27® minus insulin (A1895601, Life Technologies) (RPMI + B27-). To this medium, various small molecules were added over a week-long timetable as previously described. On the first day (D0) of differentiation, 6 µM CHIR 99021 (C-6556, LC Laboratories) was added. On D2, media was aspirated and replaced with RPMI + B27-. On D3, media was aspirated and replaced with 5 µM of IWR-1 (S7086, Selleck Chemicals) in RPMI + B27-. Media was replaced with RPMI + B27- on D5 and RPMI plus B27 supplemented with insulin (17504-044, Life Technologies) (RPMI + B27+) on D7.

Growth protocol

Pluripotent cells were grown on Matrigel-coated plates (1:100 Matrigel in E8 medium) using chemically defined E8 medium. The medium was changed daily and cells were passaged every 3-4 days using EDTA or Accutase. Cardiomyocytes were maintained in RPMI + B27+ with media change every other day.

Extracted molecule

total RNA

Extraction protocol

For RNA-seq, RNA was isolated using miRNeasy kit (Qiagen) following the manufacture’s protocol. For ATAC-seq, 100,000 cells were centrifuged 500 g for 5 min at room temperature. The cell pellet was resuspended in 50 ml lysis buffer (10 mM Tris–Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Igepal CA-630) and centrifuged immediately 500 g for 10 min at 4°C. The cell pellet was resuspended in 50 ml transposase mixture (25 ul 2×TD buffer, 22.5 μl dH2O, and 2.5 μl Illumina Tn5 transposase or with final concentration of 100 nM Atto-590-labeled in-house Tn5) and incubated at 37°C for 30 min. After transposition, the mixture was purified with the Qiagen Mini-purification kit and eluted in 10 ul Qiagen EB elution buffer. For transgenic BANCR embryos plasmid vector construction (pRP[Exp]-BetaMHC>hBANCR) and purification, pronuclear plasmid injection of FVB embryos, harvesting and genotyping of E16 embryos were performed by Cyagen Biosciences (Santa Clara, CA,USA). Total RNA was isolated from extracted embryo hearts that were plasmid positive (n=2) or negative (n=2).
Total RNA was submitted to a core facility (Stanford Functional Genomics) for library preparation and Illumina sequencing. For ATAC-seq, sequencing libraries were prepared following the original ATAC-seq protocol (Buenrostro, J. D. et al. Nat Methods 10, 1213-1218 (2013)). The HiChIP protocol was performed as previously described (Mumbach, M. R. et al. Nat Methods 13, 919-922 (2016)).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

abundance data: BOE.SCR.BKD.read.counts.txt

Data processing

ATAC-Seq reads alignment using Bowtie v2.2.6
Sort/remove duplicates using Sambamba v0.6.5
Convert to bam using Samtools v1.2.0
Count feature coverage around features using Deeptools v2.0.0
Count feature coverages using Rsubread v1.26.1
Trim reads/ quality control using Trim Galore v0.4.2
RNA-Seq reads alignment using STAR v2.5.3a
Count feature coverage around transcriptomic features using fetureCounts v1.5.0
Count feature coverages using igvtools v2.3.91
Detect differential regions using DESeq2 v1.16.1
Genome_build: hg19/panTro4/gorGor3/rheMac3/mm9
Supplementary_files_format_and_content: TDF format, read coverage

Submission date

Mar 16, 2018

Last update date

Aug 11, 2020

Contact name

Lei Tian

E-mail(s)

[email protected]

Phone

6502388262

Organization name

Stanford's Postdoctoral Scholar programs