Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
HEK293T
antibody
None

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
KAPA Hyper Prep Kit (KK8502, KapaBiosystems) For chromatin PAR-CLIP, chromatin extraction was performed based on a published protocol (Brugiolo et. al., Nucleic Acids Research, 2017, 45(18):10452–65). The chromatin pellet from 3* 15 cm of HEK293T cells was solublized 900 µL chromatin extraction buffer (20 mM Tris-Cl [pH 7.5], 100 mM KCl, 2 mM MgCl2, 2.5 mM CaCl2, 0.3 M sucrose, and 0.1% v/v Triton X-100, 1% protease and phosphatase inhibitor) with 5 U/mL Micrococcal Nuclease (N3755, Sigma) by shaking at 4 ºC for 2 hours. The immunoprecipitation was performed with 5 ug of rabbit anti-hnRNPG antibody (Ab190352, abcam) overnight and 100 uL Protein A beads (10002D, Thermo) for another 4 hours at 4ºC. Co-immunoprecipitated RNA fragments were then washed, end-repaired on-beads, size-selected by SDS-PAGE, and released by Protease K digestion. RNA was purified by Acid-Phenol:Chloroform extraction and overnight ethanol precipitation. For ChIP-seq, we followed the protocol of ChIP-IT® Express kit (53008, Active motif) with Spike-in normalization strategy. The chromatin samples were sheared by sonication into 200-500 bp in size. For each immunoprecipitation reaction, 20 ug of the sample chromatin, 5 ug of the antibody, 10 ng of Spike-in chromatin (53083, Active motif), and 1 ug of Spike-in antibody (61686, Active Motif) were used. After washing, reverse crosslinking, and treatment with RNase A and Protease K, the DNA was purifed by with DNA Clean & Concentrator-5 (D4013, Zymo research).

Sequencing Platform

instrument_model
Illumina HiSeq 4000

hg38

Number of total reads
30599013
Reads aligned (%)
98.3
Duplicates removed (%)
5.2
Number of peaks
812 (qval < 1E-05)

hg19

Number of total reads
30599013
Reads aligned (%)
97.9
Duplicates removed (%)
6.0
Number of peaks
872 (qval < 1E-05)

Base call quality data from DBCLS SRA