Antigen

Antigen Class

Histone

Antigen

H3K27ac

Cell type

Cell type Class

Blood

Cell type

Erythroblasts

MeSH Description

Immature, nucleated ERYTHROCYTES occupying the stage of ERYTHROPOIESIS that follows formation of ERYTHROID PRECURSOR CELLS and precedes formation of RETICULOCYTES. The normal series is called normoblasts. Cells called MEGALOBLASTS are a pathologic series of erythroblasts.

Sample

source_name

Cultured erythroblast from bone marrow

cell type

Cultured erythroblast from bone marrow

add-back of nsd1

none

genotype

NSD1-/-

growth_medium

Maintainance

time

0h

antibody

H3K27ac (Abcam ab4729)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Pull-down beads were Protein G sepharose (Sigma-Aldrich, P3296). Washes were done with the low salt, high salt, li-Cl wash buffers according to EZ-Magna CHIP A/G kit protocol from Millipore. DNA elution was done with the IPURE Kit V2 (Diagenode, Cat.No. C03010012). Antibodies: GATA1 ab11862 10 ug per ChIP Abcam; H3K36me3: ab9050-11 10 ug per ChIP Abcam; H3K27ac: ab4729 10 ug per ChIP Abcam ChIP protocol was adapted from the EZ-Magna ChIPTM A/G kit protocol (Millipore, Merck KGaA, Darmstadt, Germany). Cells (Nsd1 WT and Nsd1 Set in maintenance as well as 24h differentiation) were fixed with 1% formaldehyde, lysed with Cell Lysis and then Nuclear Lysis buffers to a concentration of 20^106 cells per mL, and finally sonicated (20-min cycle on Covaris apparatus; KBioscience). Sheared chromatin was immunoprecipitated overnight using the following antibodies: anti-GATA1 (Abcam, ab11852), anti-H3K36me3 (Abcam, ab9050-100), anti-H3K27ac (Abcam, ab4729). 1/10 of the sheared chromatin was used as a reference (Input). Immune complex collection was performed using Protein G Sepharose (Sigma-Aldrich, P3296) for 1h30 at 4°C. Rinses were done according to Magna ChIPTM kit protocol with Low salt, High salt and LiCL immune complex wash buffers. Finally, elution was performed according the IPure Kit protocol (Diagenode, Cat.No. C03010012) following manufacturer's instructions. Two independent ChIP-seq experiments were conducted for Gata1, H3K27ac and H3K36me3.

Sequencing Platform

instrument_model

Illumina NovaSeq 6000

mm10

Number of total reads

91896000

Reads aligned (%)

91.2

Duplicates removed (%)

19.0

Number of peaks

22670 (qval < 1E-05)

mm9

Number of total reads

91896000

Reads aligned (%)

91.0

Duplicates removed (%)

19.4

Number of peaks

23019 (qval < 1E-05)