Antigen

Antigen Class

Histone

Antigen

H3K36me3

Cell type

Cell type Class

Neural

Cell type

Hypothalamus

MeSH Description

Ventral part of the DIENCEPHALON extending from the region of the OPTIC CHIASM to the caudal border of the MAMMILLARY BODIES and forming the inferior and lateral walls of the THIRD VENTRICLE.

Sample

source_name

Hypothalamus

tissue

Hypothalamus

age

~14 weeks

genotype

Dnmt3a_[delta]D329A

antibody

H3K36me3 (Diagenode C15410192)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

ChIP-seq: Hypothalamus, liver, pituitary or epiblast was collected and flash frozen in liquid nitrogen. RNA-seq: Hypothalamus was collected and flash frozen in liquid nitrogen, when need RNA was extracted using TRIzol (Invitrogen) and cleaned using RNA Clean & Concentrator (Zymo research) and Ribo-Zero rRNA removal kit (Illumina). BiSulfite-seq: Hypothalamus or epiblats was collected and flash frozen in liquid nitrogen. Hypothalamus DNA was extracted using DNeasy Blood & Tissue kit (Qiagen), epiblast was first permeabilised and digested using micrococcal nuclease (New England Biolans) and then purified using SPRI beads. ChIP-seq: 2.5% of a whole hypothalamus was permeabilised and digested using micrococcal nuclease (New England Biolabs), and precleared using Dynabeads Protein A/G beads (ThermoFisher Scientific). Antibodies for H3K4me3 (Diagenode C15410003), H3K27me3 (Millipore 07-449), or H3K36me3 (Diagenode C15410192) were pre-bound to Protein A/G beads and incubated with chromatin for immunoprecipitation. Bound DNA was purified by SPRI purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific). MicroPlex Library Preparation kit v2 (Diagenode) was used as per manufacturer's instructions to generate libraries of immunoprecipitated samples. RNA-seq: Libraries were generated using NEBNext Ultra RNA library preparation kits (New England Biolabs) as per manufacturer's instructions. BiSulfite-seq: Imprint DNA modification kit (Sigma) one-step procedure was used to bisulphite convert the DNA. First strand synthesis was done using Klenow exo- (New England Biolabs) and biotin-tagged Illumina 9bp random sequence adaptor, followed by Exo-I treatment (New England Biolabs) and SPRI purification. Biotinylated DNA was captured using Dynabeads Strepatavidin M-280 beads (ThermoFisher Scientific) and second strand synthesis was carried out using reverse Illumina 9bp random sequence adaptor, followed by library amplification using Phusion HF (Thermo Scientific) or KAPA HiFi (Kapa Biosystems) for 10-16 cycles.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

28531860

Reads aligned (%)

98.6

Duplicates removed (%)

34.5

Number of peaks

115 (qval < 1E-05)

mm9

Number of total reads

28531860

Reads aligned (%)

98.5

Duplicates removed (%)

34.6

Number of peaks

96 (qval < 1E-05)