Antigen

Antigen Class

Unclassified

Antigen

Unclassified

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

treatment

no fear

strain background

C57BL/6

genotype

Top3b KO

tissue

brain

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Mouse brain tissue was homogenized in 1 ml of 1% formaldehyde for 15 min. The sample was added of 50 ml of 2.5M glycine (final concentration 125 mM), incubated at room temperature for 5min, and centrifuged at 1,000 x g for 5 min at 4°C. The pellet was collected and washed with PBS. The pellet was then resuspended in 400 ml of RIPA buffer with protease inhibitor cocktail and 0.5 mM phenylmethylsulfonyl fluoride and centrifuge at 1,000 x g for 10 min at 4°C. The lysates were sonicated to shear the DNA to lengths between 200 and 400 bp, and were centrifuged at 1,000 x g for 10 min at 4°C. In order to pre-clear the chromatin, 100 ml of Protein A beads were added to the sample and incubated for 60 min at room temperature. During preclearing step, Protein A beads were incubated with blocking solution (0.5% BSA in PBS) for 60 min at room temperature. 5 ml of antibody and 20-30 ml of Protein A beads were added into the precleared lysate and incubated for 4 hrs at 4C and mixed on a rotator during all the incubation step. Each sample was washed one-time with 0.3 M NaCl/RIPA buffer, one-time with RIPA buffer, one-time with 0.25 M LiCl and twice with TE. The beads were incubated for 8 hrs at 65C with 100 ml TE, 3 ml of 10% SDS and 3ml of Protease K in a thermocycler, and the liquid was transferred into a new tube. 100 ml of TE was added to the beads, resuspended, and the supernatant was transferred again to the tube in order to reach a final volume of 200 ml. ChIP-seq libraries were prepared for sequencing using standard Illumina protocols

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

38356853

Reads aligned (%)

97.6

Duplicates removed (%)

70.1

Number of peaks

16850 (qval < 1E-05)

mm9

Number of total reads

38356853

Reads aligned (%)

97.4

Duplicates removed (%)

70.2

Number of peaks

16861 (qval < 1E-05)