Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
46C mES cells
sample type
embryonic stem cells (RA, day1)
treatment
LIF withdrawal, 2uM RA, day1
strain
129/Ola
chirp probes
ChIRP probes targeting Haunt mRNA
genotype
wild type
cell line
46C
cell type
embryonic stem cells
chip antibody
none
barcode
TCACGTT

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIRP was done as described previously with the following modifications (Chu et al. 2011). First, 59 nt DNA probes were biotinylated through terminal transferase (New England Biolabs). Intensive crosslinking of cells was performed in the following sequence: two rounds of 3000J UV treatment on ice, 0.8% of formaldehyde (FMA) for 10 minutes, 2mM of DSP (Dithiobis [succinimidyl propionate]) for 30 minutes, and followed by 3.7% of FMA for 10 minutes at room temperature. Crosslinked cell pellets were lysed. Chromatin was fragmented into a range of 2 - 5 kb by sonication. Hybridization, washing and elution steps were performed as described previously, except that we included an additional stringent wash (0.1x SSC). After elution and reverse crosslinking, RNA was further extracted by TRIzol reagent (Life Technologies). The library was constructed by library preparation modules (New England Biolabs) according to manufacturer's instructions. Briefly, ChIRP retrived RNA was fragmentated by NEBNext Magnesium RNA Fragmentation Module at 94 ℃ for 210 seconds. The fragementated RNA was reverse transcribed by SuperScript III First-Strand Synthesis System for RT-PCR kit (Life Technology), 2'nd strand was then synthesized by NEBNext mRNA Second Strand Synthesis Module , the end repair, dA-tailing and adaptor ligation were also following the instruments by respect module. the adaptor for the libary was sythersized from IDT with the first 7nt is "TCACGTT" as a barcode. After liagation, the DNA was amplified by primer 1.0 (AATGATACGGCGACCACCGAGATCTACAC, synthersized from IDT) and 2.0 (CAAGCAGAAGACGGCATACGAGAT, synthersized from IDT) for 15 cycles. After this, the product was further purified and size selected by Ampure XP beads (Beckman Coulter). The library was sequenced on the Genome Analyzer following the manufacturer's protocols. Total RNA of RNA-Seq were extracted by TRIzol reagent according to manufacturer's instruction. mRNA were further isolated by Dynabeads mRNA purification kit (Life Technologies) according to manufacturer's instruction. Libraries were prepared by illumina TruSeq Stranded mRNA LT Sample Prep Kit (RS-122-2101) according to the standard protocols of the manufacturer. ChIRP retrieved DNA-seq were prepared by library preparation modules (New England Biolabs) according to manufacturer's instructions.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
12675388
Reads aligned (%)
95.8
Duplicates removed (%)
52.3
Number of peaks
165 (qval < 1E-05)

mm9

Number of total reads
12675388
Reads aligned (%)
95.7
Duplicates removed (%)
52.5
Number of peaks
136 (qval < 1E-05)

Base call quality data from DBCLS SRA