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| Status |
Public on Jul 12, 2024 |
| Title |
6b36c7f9-4084-4763-b6ca-a54bb9dfc6c6 |
| Sample type |
SRA |
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| Source name |
iPSC-derived cardiovascular progenitors
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| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC-derived cardiovascular progenitors
treatment: IFNg
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| Treatment protocol |
On day 28 (three days after thawing), CVPCs were treated with 100 nM human recombinant IFN-γ (ThermoFisher) for 24h. On day 29, control and IFN-γ-treated CVPC samples (eight in total) were harvested.
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| Growth protocol |
iPSC lines from four unrelated individuals recruited as part of the iPSCORE project were previously differentiated into iPSC-CVPCs using small molecules and enriched using metabolic selection with lactate (Table S1). The CVPCs were harvested at day 25, which we and others have previously shown represent fetal cardiac cells, and cryopreserved at -80° C. The cryopreserved CVPCs (day 25) were then thawed, plated at a density of 2.5x105/cm2 cells on Matrigel (Corning) coated 6-well plates, and allowed 72 hours for recovery and expansion.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA-seq: We extracted total RNA from lysed pellets frozen in RLT Plus buffer (collected on day 29) using the Quick-RNA™ MiniPrep Kit (Zymo Research), assessed quality to make sure RNA Integrity number (RIN) was 7.5 or greater. ATAC-seq: ATAC-seq samples were processed as previously described in detail25. Briefly, frozen nuclear pellets of 1x105 CVPC were thawed on ice and tagmented in total volume of 25μl in permeabilization buffer containing digitonin (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.01% digitonin) and 2.5μl of Tn5 from Nextera DNA Library Preparation Kit (Illumina) for 45-75min at 37°C in a thermomixer (500 RPM shaking).
RNA-seq: We prepared indexed libraries using the Illumina TruSeq stranded mRNA kit and sequenced with 150 bp paired-end reads on an Illumina HiSeq 4000 (mean = 91.4M reads). ATAC-seq: To eliminate confounding effects due to index hopping, all libraries within a pool were indexed with unique pairs of i7 and i5 barcodes. Libraries were amplified for 12 cycles using NEBNext® High-Fidelity 2X PCR Master Mix (NEB) in total volume of 25µl in the presence of 800nM of barcoded primers (400nM each) custom synthesized by Integrated DNA Technologies (IDT) and sequenced with 150-bp paired end reads on a HiSeq4000 (mean = 112.9M reads).
One RNA-seq and ATAC-seq library was generated for each sample. Four CVPCs with 2 treatments = 8 RNA-seq and 8 ATAC-seq samples.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
ATAC-seq for UDID088
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| Data processing |
RNA-seq: To measure gene expression in the 8 CVPC samples, we obtained raw counts in transcript per million bp (TPM) as previously described. Briefly, FASTQ files were aligned to the hg19 reference human genome using with STAR v.2.7.3 and GencodeV.34lift37 with parameters: --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000. Next, the BAM files were sorted and indexed using sambamba v0.6.7, and duplicates were marked using bammarkduplicates from the biobambam software. To quantify gene expression (TPM), we used RSEM version 1.2.20 with the following parameters: rsem-calculate-expression –bam --estimate-rspd --paired-end --forward-prob 0. RNA-seq sample quality was analyzed with samtools stats, idxstats, and Picard RNA Metrics.
ATAC-seq: The 16 FASTQ files were aligned to the hg19 reference genome with STAR using the following parameters; --outFilterMultimapNmax 20, -- outFilterMismatchNmax 999, --outFilterMismatchNoverLmax 0.04, --seedSearchStartLmax 20, -- outFilterScoreMinOverLread 0.1, outFilterMatchNminOverLread 0.1. Sample mate coordinates were filled using samtools fixmate and duplicates were marked using samtools markdup. We then removed poorly mapped reads, fragments < 38bp, fragments > 2,000 bp, and reads that did not map to autosomal or sex chromosomes with samtools view. Finally, we removed reads in the blacklisted regions (https://mitra.stanford.edu/kundaje/akundaje/release/blacklists/hg19-human/wgEncodeHg19ConsensusSignalArtifactRegions.bed.gz) using bedtools intersect with the v parameter. Narrow peaks were called using MACS2 on each of the 8 BAM files individually, using the following parameters; -f BAMPE -g hs --nomodel --shift -100 --extsize 200 --call-summits –q 0.01. We merged the peak bed files from the 8 ATAC-seq samples, then removed peaks that were only present in one sample which resulted in 119,794 reference peaks. For each sample, we counted the number of reads in the reference peaks using featureCounts.
Assembly: hg19
Supplementary files format and content: RNA-seq: RSEM TPM measurements
Supplementary files format and content: ATAC-seq: Counts for reference narrow peaks
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| Submission date |
Apr 09, 2024 |
| Last update date |
Jul 12, 2024 |
| Contact name |
Timothy D Arthur |
| E-mail(s) |
tdarthur40@gmail.com
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| Organization name |
UCSD
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| Department |
Pediatrics
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| Street address |
9500 Gilman Drive #0761
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093-0761 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE263611 |
IFN-γ activates an immune-like regulatory network in the cardiac vascular endothelium |
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| Relations |
| BioSample |
SAMN40905119 |
| SRA |
SRX24201771 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8195221_6b36c7f9-4084-4763-b6ca-a54bb9dfc6c6.narrowPeak.gz |
2.4 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
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