Antigen

Antigen Class

TFs and others

Antigen

Mef2d

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

tissue

retina

genotype

wild type

age

postnatal day 11

chip antibody

anti-MEF2D (Flavell et al., 2008)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

MEF2D ChIP from mouse retinas was performed as previously described for brain tissue (Hong et al., 2008) with the following modifications: p11 mouse retinas were dissected in ice-cold HBSS prior to homogenization and crosslinking. 4μg of anti-MEF2D antibody was pre-bound to 15μl of Protein A dynabeads (Life Technologies) per immunoprecipitation (IP) from approximately 100 million retinal cells. H3K27Ac ChIP was performed as described above with the following modifications: 10 mM sodium butyrate was added to all solutions until post-IP washes with the exception of cross-linking buffer. Chromatin was fragmented for H3K27Ac ChIP by brief sonication followed by MNase (New England Biolabs) digestion for 8 minutes at 37C to generate mononucleosomes. 0.25μg of anti-H3K27Ac antibody was used per IP from 10 million retinal cells. After reverse crosslinking all samples were purified using phenol/chloroform/isoamyl alcohol followed by column clean up. Single-end short read sequencing libraires were prepared using standard protocols, following the manufacturers instructions. were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

20620653

Reads aligned (%)

97.9

Duplicates removed (%)

15.5

Number of peaks

333 (qval < 1E-05)

mm9

Number of total reads

20620653

Reads aligned (%)

97.8

Duplicates removed (%)

15.6

Number of peaks

328 (qval < 1E-05)