Antigen

Antigen Class

Unclassified

Antigen

Unclassified

Cell type

Cell type Class

Blood

Cell type

Hematopoietic Stem Cells

MeSH Description

Progenitor cells from which all blood cells derived. They are found primarily in the bone marrow and also in small numbers in the peripheral blood.

Sample

source_name

sorted murine lin-/Sca-1/c-Kit in vitro cultured cells

strain

C57Bl/6.SJL congenic

cell type

LSK hematopoietic stem cells

treatment

4-OHT 48 hrs

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cells in 96 wells were pooled and resuspended to 1 million/mL in phosphate-buffered saline (PBS) containing 1% formaldehyde and incubated 10 minutes, followed by centrifugation and resuspension in 50 mM glycine/PBS, a 10 minute incubation, then a PBS incubation for 10 minutes. All steps were performed at room temperature at a cell density of 1 million/mL. Fixed cell pellets were either processed immediately or stored at -80°C.To shear the chromatin, the cell pellet was resuspended in lysis buffer (Tris pH 7.5, EDTA 1 mM, SDS 1%, 1X protease inhibitor complex, Roche) at 5x104 cells per 20 µL. Low-retention surface barrier tips were used for all steps (CLP Neptune). Sonication was performed for 10 cycles (30 seconds with 30 seconds rest) using a Bioruptor UCD-200 (Diagenode Inc.). Sonicated chromatin was centrifuged at 13,000 x g at 4°C for 5 minutes and the supernatant was diluted 10-fold with 2X RIPA buffer (20 mM Tris pH7.5, 2 mM EDTA, 2% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate, 200 mM NaCl). For each ChIP reaction 200µL diluted chromatin incubated with 1 µg antibody overnight at 4°C then 7.5 µL each of protein A and protein G Dynabeads (Invitrogen), previously washed in 1X RIPA buffer, were added to each immunoprecipitation and incubated for additional 2 hours at 4°C. The bead:protein complexes were washed three times with 200 µL 1X RIPA buffer and once with 200 µL TE (10mM Tris pH 7.5, 1mM EDTA). Genomic DNA was eluted from the ChIP and input samples for 3 hours at 65°C in 300 µL elution buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 µg/mL proteinase K) using an Eppendorf Thermomixer at 1,000 rpm. Samples were phenol/chloroform extracted, ethanol precipitated with 10 µg each linear acrylamide and glycogen then centrifuged at 13,000xg for 20 minutes at 4°C. Pellets were air dried and resuspended in 15 µL TE containing 0.1 mM EDTA. The library for sequencing was constructed using Ovation Ultralow DR Multiplex System 1-8 according to manufacturer's instructions (Nugen, San Carlos CA). Libraries were sequenced using HIseq-2000 (Illumina, San Diego, CA) to obtain 50 bp long reads. Half of the input or of the ChIP sample was used to prepare sequencing libraries with specific barcodes for individual ChIP sample according to the manufacturer’s instructions (Ovation Ultralow Library Systems, NuGEN). DNA concentration in each ChIP-library was measured using Qubit Fluorometer (Invitrogen) and 10 nM DNA from each library was used for running next-generation multiplexed sequencing using 50 bp single-end reads on a HiSeq-2000 instrument (Illumina).

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

35132609

Reads aligned (%)

97.0

Duplicates removed (%)

15.3

Number of peaks

549 (qval < 1E-05)

mm9

Number of total reads

35132609

Reads aligned (%)

96.7

Duplicates removed (%)

15.2

Number of peaks

600 (qval < 1E-05)