Antigen

Antigen Class

Histone

Antigen

H3K9me3

Cell type

Cell type Class

Neural

Cell type

Retinal Pigment Epithelium

MeSH Description

The single layer of pigment-containing epithelial cells in the RETINA, situated closely to the tips (outer segments) of the RETINAL PHOTORECEPTOR CELLS. These epithelial cells are macroglia that perform essential functions for the photoreceptor cells, such as in nutrient transport, phagocytosis of the shed photoreceptor membranes, and ensuring retinal attachment.

Sample

source_name

RPE

tissue

retinal pigment epithelium (RPE)

strain

C57BL/6

chip antibody

H3K9me3 (Diagenode Cat# C15410193)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Freshly isolated RPE 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 14,000-28,000 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

83719421

Reads aligned (%)

97.5

Duplicates removed (%)

57.1

Number of peaks

757 (qval < 1E-05)

mm9

Number of total reads

83719421

Reads aligned (%)

97.0

Duplicates removed (%)

57.1

Number of peaks

915 (qval < 1E-05)