Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Neural

Cell type

Retina

MeSH Description

The ten-layered nervous tissue membrane of the eye. It is continuous with the OPTIC NERVE and receives images of external objects and transmits visual impulses to the brain. Its outer surface is in contact with the CHOROID and the inner surface with the VITREOUS BODY. The outer-most layer is pigmented, whereas the inner nine layers are transparent.

Sample

source_name

retina

strain

C57BL/6J

age

P28

genotype

Nrlp-GFP

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Flow-sorted cells were lysed using lysis buffer and sonicated using Covaris. Cellular debris was removed by centrigugation, and clear chromatin solution was reconstituted to RIPA buffer. Pre-cleared chromatin solution was then incubated with anti-H3K4me3 (ab8580, Abcam, MA, USA), anti-H3K27me3 (07-449, Millipore, MA, USA) or normal IgG (12-370, Millipore) with constant rotation at 4°C overnight. Thoroughly washed immunoprecipitation complexes were then digested, and the cross-link was reversed by incubation in 50 l of TE, pH 8 supplemented with 0.3% SDS and 1 mg/ml proteinase K at 65 °C for 6 hrs. The carried-over RNA was digested by 1 hr incubation at 37°C with RNase cocktail (Ambion, Life Technologies), and the immunoprecipitated DNA was then purified using Agencourt AMPure XP beads (Beckman Coulter, CA, USA). Libraries were prepared using TruSeq DNA Sample Preparation kit v2 (Illumina, FC-121-2001; FC-121-2002) according to Illumina's instructions with the following modifications. 1. As amount of total ChIP DNA obtained from each ChIP experiment is estimated to be only 400 pg to 1 ng, entire ChIP DNA was used for ChIP-seq library construction Instead of 1 ug DNA as recommended by Illumina. 2. For adapter ligation, 1/100 dilution of adapter was used to avoid having access adapter-adapter dimer. 3. After adapter ligation, double-sided SPRI technique (200-500 nt fragments were selected with 0.55x left /0.75x right) was used for size-selection in order to minimize a sample loss. 4. 18 cycles of PCR amplification were performed on all ChIP-seq libraries.

Sequencing Platform

instrument_model

Illumina Genome Analyzer IIx

mm10

Number of total reads

41935709

Reads aligned (%)

96.5

Duplicates removed (%)

79.7

Number of peaks

2797 (qval < 1E-05)

mm9

Number of total reads

41935709

Reads aligned (%)

95.7

Duplicates removed (%)

79.6

Number of peaks

3002 (qval < 1E-05)