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Sample GSM5570815

Query DataSets for GSM5570815

Status

Public on Feb 03, 2022

Title

PROseq_auxin_2hr_N4

Sample type

SRA

Source name

Ramos cell

Organism

Homo sapiens

Characteristics

treatment: Two Hour 100 uM Indole-3-Acetic Acid

Treatment protocol

Cells were treated with 100 uM IAA or 500 nM C16, and 0.1% DMSO for the times indicated.

Growth protocol

Ramos cells were obtained from ATCC (CRL-1596) and cultured with the recommended conditions (RPMI media supplemented with L-glutamine, 10% FBS, 100 IU/ml Penicillin, and 100 μg/ml streptomycin).

Extracted molecule

nuclear RNA

Extraction protocol

RNA-seq: 5 x 10^6 cells per sample were lysed using TRIZOL. The RNA was phase separated with chloroform, precipitated with isopropyl alcohol, and DNase treated .
ChIP-seq:ChIP-seq: Cells were resuspended in PBS at 4x106 cells/ml, and crosslinked with 1% methanol free formaldehyde for 10 min at room temperature before quenching with 125 mM Glycine. Cells were pelleted, washed twice with 1.5 ml PBS, and lysed in lysis buffer (50 mM Tris pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% SDS and protease inhibitor cocktail) at a concentration of 80x106 cells per ml. Aliquots of 300 ul were sonicated in a Bioruptor for 20 min and debris was removed by centrifugation. 100 ul of Chromatin was diluted to 1 ml using lysis buffer lacking SDS and incubated overnight at 4C with the appropriate antibody or control IgG. Antibodies were captured on 15 ul bed volume Protein A agarose beads and immune complexes were washed with 1 ml Low Salt Buffer (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton), 1 ml Hi Salt Wash Buffer (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton), 1 ml LiCl Wash Buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Triton) and twice with 1 ml of TE. Samples were decrosslinked overnight in 100 ul TE containing 20 ug of Proteinase K (PK) at 65C then brought up to 500 ul with water and incubated at 95C for 20 minutes to inactive PK.
PRO-seq: Nuclei from 30 x 10^6 Ramos cells per sample were isolated in buffer using dounce homogenization then flash frozen and kept at -80C until run on reaction. Primary transcripts were eluted using TRIZOL extractions and dynabeads, which isolate primary transcripts by binding the biotin incorperated during the run-on.
RNA-seq: Library preparation with rRNA depletion performed by Vanderbilt Technologies for Advanced Genomics (VANTAGE)
PRO-seq: RNA was used in a reverse transcriptase reaction to generate a cDNA library. Libraries were amplified based on the a PCR cycle number determined from a test analysis of a portion of sample. Library amplification was performed with Illumina-based index primers.
ChIP-seq: ChIP-seq: After de-crosslinking, samples were purified using the MinElute PCR Purification Kit (Qiagen). The NEBNext Ultra II DNA Library Prep Kit for Illumina was used to prepare unique dual indexed libraries from the entire eluate following the manufacture’s protocol.
RNA-seq, ChIP-seq, PRO-seq

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

PROseq_count_pp_gb.txt

Data processing

RNA-seq reads were aligned to the hg19 genome assembly using STAR after adapter trimming, and quantified by featureCounts.
Differential analysis were performed by DESeq2.
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie2.
Peaks were called by MACS2 with a q-value of 1e-5.
Differential binding peaks were identified using DiffBind.
After adapter trimming and low quality sequence removal by cutadapt, PRO-seq reads longer than 15bp were reversed complemented using FastX tools.
Reverse complements of the trimmed reads were aligned to the human genome hg19 using Bowtie2.
Reads mapped to rRNA loci and reads with mapping quality less than 10 were removed.
Bam files were given to NRSA (http://bioinfo.vanderbilt.edu/NRSA/) to estimate RNA polymerase abundance in proximal-promoter and gene body regions of genes, to calculate pausing index and pausing index alterations, and to detect enhancers and quantify eRNA changes.
Genome_build: hg19
Supplementary_files_format_and_content: RNA-seq: tab-delimited text file include raw counts for each Sample
Supplementary_files_format_and_content: ChIP-seq: one peak file per sample
Supplementary_files_format_and_content: PRO-seq: tab-delimited text file include raw counts for proximal-promoter and gene body region, and density for gene body

Submission date

Sep 09, 2021

Last update date

Feb 03, 2022

Contact name

Jing Wang

Organization name

Vanderbilt University Medical Center

Street address

2220 Pierce Avenue

City

Nashville