Antigen

Antigen Class

RNA polymerase

Antigen

RNA polymerase II

Cell type

Cell type Class

Neural

Cell type

Brain

MeSH Description

The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM.

Sample

source_name

brain

mouse strain

CD1

Stage

P1

antibody

PolII (Santa Cruz, sc899, lot#, G2414)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP-seq in mouse brain, the whole brains were homogenized with a glass douncing homogenizer. The cell homogenate was fixed with 1% paraformaldehyde for eight min at room temperature and quenched with 0.125 M glycine for five min at room temperature, followed by centrifugation through a 29% iodixanol cushion. The nuclei pellet was resuspended and sonicated using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Sepharose, chromatin was incubated with antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with the same BSA-blocked protein G beads.For ChIP-seq in 393T-REx cells, cells were crosslinked with 1% formaldehyde for eight min at room temperature and the nuclei were then isolated. As described above, following sonication and pre-clearing, chromatin IP was performed to isolate associated DNA. For ChIP-seq library construction, 5-10 ng of DNA extracted from the chromatin immunocomplexes. Libraries were prepared according to manufacturer’s instructions (Illumina). Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), and ligated to custom adapters with LigaFast (Promega, city, state #M8225). Fragments of 350±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530).

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

23052606

Reads aligned (%)

93.1

Duplicates removed (%)

35.6

Number of peaks

10264 (qval < 1E-05)

mm9

Number of total reads

23052606

Reads aligned (%)

92.9

Duplicates removed (%)

35.7

Number of peaks

10268 (qval < 1E-05)