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Sample GSM3109156

Query DataSets for GSM3109156

Status

Public on Aug 09, 2018

Title

317155

Sample type

SRA

Source name

coronary artery smooth muscle cell

Organism

Homo sapiens

Characteristics

sample type: cell culture
cell line: 317155

Treatment protocol

All cell lines were cultured in smooth muscle growth medium (Lonza catalog # CC-3182) supplemented with hEGF, insulin, hFGF-b, and 5% FBS, according to Lonza instructions. All HCASMC lines were expanded to passage 5-6 prior to extraction.

Growth protocol

Sample acquisition and cell culture. A total of 52 primary human coronary artery smooth muscle cell (HCASMC) lines collected from donor hearts were purchased from PromoCell (catalog # C-12511, n = 19), Cell Applications (catalog # 350-05a, n = 25), Lonza (catalog # CC-2583, n = 3), Lifeline Cell Technology (catalog # FC-0031, n = 3), and ATCC (catalog # PCS-100-021, n = 2). All lines were stained with smooth muscle alpha actin to check for smooth muscle content and all lines tested negative for mycoplasma (Table S1).

Extracted molecule

total RNA

Extraction protocol

Library preparation and sequencing. RNA was extracted using Qiagen miRNeasy Mini Prep Kit (catalog # 74106). Quality of RNA was assessed on the Agilent 2100 Bioanalyzer. Samples with RIN greater than or equal to 8 were sent to the Next-Generation Sequencing Core at the Perelman School of Medicine at the University of Pennsylvania. Libraries were made using Illumina TruSeq Stranded Total RNA Library Prep Kit (catalog # 20020597) and sequenced using 125bp paired-end on HiSeq 2500 Platform. We used ATAC-seq to assess chromatin accessibility with slight modifications to the published protocol {Buenrostro:2015be}. Approximately 5e4 fresh cells were collected at 500 g, washed in PBS, and nuclei extracted with cold lysis buffer. Pellets were subjected to transposition containing Tn5 transposases (Illumina) at 37 °C for 30 min, followed by purification using the DNA Clean-up and Concentration kit (Zymo). Libraries were PCR amplified using Nextera barcodes, with the total # of cycles empirically determined using SYBR qPCR. Amplified libraries were purified and quantified using bioanalyzer, nanodrop and qPCR (KAPA) analysis. Libraries were multiplexed and 2x75 bp sequencing was performed using an Illumina NextSeq 500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

rnaseqc.hcasmc_eqtl.reads.gct

Data processing

Alignment and quantification of transcriptomic and epigenomic features. De-multiplexed FASTQ files were mapped with STAR version 2.4.0i in 2-pass mode{Dobin:2013fg} over the hg19 reference genome. Prior to expression quantification, we filtered our reads prone to mapping bias using WASP {vandeGeijn:2014wg}. Total read counts and RPKM were calculated with RNA-SeQC v1.1.8{Deluca:2012dp} using default parameters with additional flags “-n 1000 -noDoC -strictMode” over GENCODE v19 reference. Allele-specific read counts were generated with the createASVCF module in RASQUAL{Kumasaka:2016fn}. We quantified intron excision levels using LeafCutter intron-spanning reads{Li:2016wz}. In brief, we converted bam files to splice junction files using the bam2junc.sh script, and defined intron clusters using leafcutter_cluster.py with default parameters, which requires at least 30 reads supporting each intron and allows intron to have a maximum size of 100kb. We used the ENCODE ATAC-seq pipeline to perform alignment and peak calling(https://github.com/kundajelab/atac_dnase_pipelines){Sloan:2016hy}. FASTQ files were trimmed with Cutadapt v1.9{Martin:2011va} and aligned with Bowtie2 v2.2.6{Langmead:2012jh}. MACS2 v2.0.8 {Zhang:2008gm} was used to call peaks with default parameters. Irreproducible Discovery Rate (IDR){Li:2011gg} analyses were performed based on pseudo-replicates (subsample of reads) with a cutoff of 0.1 to output an IDR call set, which was used for downstream analysis. We used WASP {vandeGeijn:2014wg} to filter out reads that are prone to mapping bias.
Genome_build: hg19

Submission date

Apr 23, 2018

Last update date

Aug 09, 2018

Contact name

Thomas Quertermous

E-mail(s)

tomq1@stanford.edu

Phone

650-723-5012

Organization name

Stanford University