Antigen

Antigen Class

Histone

Antigen

H3K27me3

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

Glast+ cells from P28 retinas

tissue

retina

strain

C57BL/6

cell type

Glast+ cells

chip antibody

H3K27me3 (Diagenode Cat# C15410195)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Freshly isolated cells were cross-linked in 1% formaldehyde with 1X PBS. To stop the cross linking reaction, glycine was added to a final concentration of 0.125M. The chromatin was prepared using the True MicroChIP Kit (Diagenode Cat# C01010130). Chromatin was sheared using the Bioruptor® Pico sonication device (Diagenode Cat# B01060001). ChIP was performed using IP-Star® Compact Automated System (Diagenode Cat# B03000002) following the protocol of the aforementioned kit. Chromatin corresponding to 25,000 cells was immunoprecipitated using the antibodies. Libraries were prepared from input and ChIP'd DNA (500 pg) using the MicroPlex Library Preparation Kit v2 (12 indices) (Diagenode Cat# C05010013). Library amplification is assessed using the High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). Libraries were then purified using Agencourt® AMPure® XP (Beckman Coulter) and quantified using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854). Finally their fragment size was analyzed by High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). When the proportion of fragments >500bp was too high, libraries were subjected to a double size selection using Agencourt® AMPure® XP (Beckman Coulter). Libraries were pooled and sequenced on an Illumina HiSeq 4000 with single-end reads of 50bp lengths, running HiSeq Control Software HD version 3.4.0.38.

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

90709208

Reads aligned (%)

97.4

Duplicates removed (%)

57.4

Number of peaks

853 (qval < 1E-05)

mm9

Number of total reads

90709208

Reads aligned (%)

97.0

Duplicates removed (%)

57.4

Number of peaks

938 (qval < 1E-05)