Antigen

Antigen Class

RNA polymerase

Antigen

RNA polymerase II

Cell type

Cell type Class

Pluripotent stem cell

Cell type

Embryoid Bodies

MeSH Description

Spontaneous aggregations of human embryonic stem cells that occur in vitro after culturing in a medium that lacks LEUKEMIC INHIBITORY FACTOR. The embryoid bodies can further differentiate into cells that represent different lineages.

Sample

source_name

E14Tga2.mESC derived Embryoid bodies

genotype

Wild type

tissue

CRL-1821 cell line

chip antibody

RNAPII, Santa Cruz, #sc-899

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP-seq, nuclei was isolated from cells fixed with 1% Formaldehyde. Next, chromatin was fragmented into ~250bp. Chromatin immunoprecipitation was performed with the indicated antibodies. For RNA-seq, total RNA was isolated with RNAeasy (Qiagen) and reverse transcribed using Superscript III and random hexamers (Life Technologies) to synthesize the 1st strand. Second strand was synthesized with dUTP to generate strand asymmetry using DNA Pol I (NEB, M0209L) and the E. coli ligase (Enzymatics, L6090L). For ChIP-seq and RNA-seq, libraries were prepared by standard protocol. First, fragments are repaired to generate blunt 5' P ends using 'EndIT DNA End Repair kit' (Epicentre). Second, 3' A overhang was generated using 'Klenow Fragment (3´→ 5´ exo-)' (NEB). Third, Illumina barcodes were ligated using 'Rapid T4 DNA Ligase' (Enzymatics). Lastly, libraries were size selected using 'Ampure beads' (Beckman Coulter), then amplified, quantified and pooled.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

41708264

Reads aligned (%)

82.3

Duplicates removed (%)

9.6

Number of peaks

10566 (qval < 1E-05)

mm9

Number of total reads

41708264

Reads aligned (%)

82.2

Duplicates removed (%)

9.6

Number of peaks

10575 (qval < 1E-05)