|
| Status |
Public on Dec 09, 2020 |
| Title |
Input_15 |
| Sample type |
SRA |
| |
|
| Source name |
Rat adult cardomyocytes
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: primary cultured rat adult ventricular cardiomyocytes
chip antibody used for chip or oligonucletides used for chirp (chromatin isolation by rna purification): Input
|
| Treatment protocol |
2.4 millions adult rat ventricular myocytes infected with 300 Multiplicity of Infection (MOI) of adenovirus for 24 hours and/or treated with 20 µM phenylephrine for 1 hour were used for each individual ChIP assay.
|
| Growth protocol |
Cells were cultured in ACCT medium (M199 Medium, Creatine 5 mM, L-carnitine 2 mM, Taurine 5 mM, HEPES 25 mM, 2,3-Butanedione monoxime 10 mM, BSA 0.2% and 1X Insulin-Transferrin-Selenium Supplement) after langendorff perfusion.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells for RNA Polymerase II (Pol II) and H3K27ac ChIPs were directly incubated with 1% formaldehyde in PBS for 10 minutes, while all other cells were subjected to cross-linking by disuccinimidyl glutarate for 30 minutes prior to 15 minutes 1% formaldehyde fixation. Cells were washed with PBS for three times, lysed in 1% SDS buffer, and then ChIP assay was performed using the EZ-ChIP kit (Millipore, 17-371), performed as recommended by the manufacturer using 1.4 - 2.9 µg antibody for each immunoprecipitation. To sequence ChIP-enriched DNA, library constructions were performed using KAPA HyperPrep Kits for NGS DNA Library Prep (Kapa Biosystems). Inputs ranged from undetectable via the Qubit dsDNA HS Assay (<10pg/µl) to 5 ng/µl. 14 PCR cycles were adjusted according to input. Samples were barcoded to allow for multiplexing and analysis on an Illumina HiSeq 4000. Cluster generation took place on the Illumina cBot according to the manufacturer's recommendations. Sequencing took place on the Illumina HiSeq 4000 using the reagents provided in the Illumina TruSeq PE Cluster and SBS kits. An average of 15 million single-end 75 base pass-filter reads were generated per sample.
To sequence ChIP-enriched DNA, library constructions were performed using KAPA HyperPrep Kits for NGS DNA Library Prep (Kapa Biosystems).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Flag-SRF(S103A) experiment 2, Input for both PolII and H3K27ac ChIPs
|
| Data processing |
Basecalls performed using CASAVA version 1.4
reads were aligned to the rn6 genome assembly using Bowtie version 2.0.1.
Genome binding peaks for SRF or phospho-Ser103 antibodies were identified using the ‘findPeaks’ command in HOMER with setting of ‘-style factor’: 200 bp peaks with 3-fold enrichment and 0.01 FDR significance over local tags.
HOMER-IDR program was employed to format the data for the IDR R package to determine the IDR threshold and identify the top peaks above that threshold.
Genome_build: rn6
Supplementary_files_format_and_content: bigWig files were generated using the ‘makeUCSCfile’ command in HOMER with setting of ‘-fsize 1e50 -o auto’.
|
| |
|
| Submission date |
Jul 24, 2019 |
| Last update date |
Dec 09, 2020 |
| Contact name |
Yuliang Tan |
| E-mail(s) |
yut020@ucsd.edu
|
| Organization name |
University of California, San Diego
|
| Department |
School of Medicine
|
| Street address |
Room 345, CMM West, 9500 Gilman Drive, Mail Code 0648
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037-0648 |
| Country |
USA |
| |
|
| Platform ID |
GPL22396 |
| Series (2) |
| GSE134799 |
ChIP assay in adult ventricular cardiomyocytes |
| GSE134801 |
Adult ventricular cardiomyocytes |
|
| Relations |
| BioSample |
SAMN12354105 |
| SRA |
SRX6581763 |
