Antigen

Antigen Class

Input control

Antigen

Input control

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

HSC_input

strain

C57BL/6

cell type

hematopoietic stem cells

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cells were fixed in 1% formalin for 10 min at room temperature and fixation was stopped by adding glycine to a final concentration of 125 mM. After three washes with PBS, cell pellets were snap-frozen and stored at -80°C. For sonication, cell pellets were resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) supplemented with protease inhibitor cocktail (Roche) and processed in a Covaris S2 instrument using empirically established conditions that resulted in chromatin fragments between 150 and 300 bp in length (Duty Cycle 5%, Intensity 4, Cycles/Burst 200; 40 cycles: 30 sec on, 20 sec off). Sonicated chromatin was cleared by centrifugation for 10 min at 13000 rpm. Ten percent of cleared chromatin was taken as input control. The H4K16ac antibody (07-329, Millipore) was coupled to protein A/G magnetic beads and washed 2x with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). Sonicated chromatin was first diluted 10-fold with ChIP dilution buffer, then the antibody-bead conjugates were added and incubated at 4°C over night. Beads were first washed 2x with Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl) and then washed 2x with LiCl Immune Complex Wash Buffer (0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1). Then precipitated chromatin was eluted and decrosslinked (2% SDS, 0.1 M NaHCO3, 0.25 M NaCl) for 4 h at 65°C and afterwards treated with proteinase K for 1 h at 45°C. DNA was purified using SPRI beads (Agencourt AMPure XP, Beckman Coulter) at a ratio of 1.8/1 (beads/DNA) and eluted in 15 µl ddH2O. Linear DNA amplification (LinDA) of ChIPed DNA was performed as described (Shankaranarayanan et al., 2011). Briefly, ChIPed DNA was dephosphorylated for 10 min at 37°C using 1 U TSAP (M9910, Promega) in NEB 4 buffer (B7004, NEB) followed by heat inactivation for 15 min at 75°C. DNA was then T-tailed for 20 min at 37°C in the presence of 5 µM T-mix (92 % dTTP, 8 % ddCTP) and 0.25 mM CoCl2 using 20 U terminal transferase (M0252, NEB) followed by heat inactivation for 10 min at 70°C. Next, 5 pmol T7-BpmI-oligo(A)14 primer (AATTAATACGACTCACTATAGGGCTGGAGAAAAAAAAAAAAAA) was annealed at 37°C for 5 min and then double strand synthesis was performed for 55 min at 37°C using 10 U Klenow fragment (M0210, NEB), 0.2 mM dNTPs. After heat inactivation, DNA was purified using SPRI beads at a ratio of 1.8/1 (beads/DNA) and eluted in 12.5 µl ddH2O. In vitro transcription was performed over night using the RNAMaxx high yield kit (200339, Stratagene) and resulting RNA was purified using SPRI beads (Agencourt AMPure XP, Beckman Coulter; beads/DNA ratio 1.8/1) followed by a reverse transcription (1h, 42°C; 10 min, 75°C) using the T7-BpmI-oligo(A)14 primer and the Superscript III reverse transcription kit (18080044, Invitrogen). Second strand synthesis was performed using a custom reaction (20 µl first strand cDNA, 10 µl 10x thermopol buffer, 1 ml 100x BSA, 3 ml 10 mM dNTPs, 0.5 µl RnaseH, 1 µl Taq polymerase, 0.1 µl Pfu polymerase, 64.4 ml H2O) which was incubated in a thermocycler under the following conditions: 37°C 5 min, 65°C 1 min, 72°C 30 min) followed by SPRI purification (beads/DNA ratio 1.8/1). Resulting double-stranded DNA was then digested with BpmI for 2 h at 37°C, purified again with SPRI beads (beads/DNA ratio 1.5/1) and eluted in 13 µl ddH2O. Illumina sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370, NEB) and the NEBNext Multiplex-Oligos for Illumina (E7335, NEB) following the manufacturer's protocol. Since for all samples, input amount of DNA was below 100 ng, the adaptors were diluted 1:10 in sterile water before adaptor ligation. Cleanup of adaptor-ligated DNA was performed without size selection using a 1.0/1 ratio of SPRI beads/DNA. For the PCR amplification of libraries, 0.5 µl of 100x Syber Green was added to each reaction and the PCR amplification was performed on a LightCycler 480 (Roche) using the following cycling conditions: initial denaturation (98°C, 30 sec) followed by 9-12 cycles (98°C, 10 sec; 65°C, 30 sec; 72°C, 30 sec). The PCR reaction was stopped, when the amplification curves had reached a fluorescence intensity of about 10. The PCR reactions were purified using SPRI beads at a ratio of 1.0/1 (beads/DNA). Sequencing libraries were multiplexed and subjected to 50 bp single-end sequencing on an Illumina HiSeq 2000 instrument (Illumina Inc., San Diego).

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

316655860

Reads aligned (%)

77.6

Duplicates removed (%)

29.9

Number of peaks

22747 (qval < 1E-05)

mm9

Number of total reads

316655860

Reads aligned (%)

77.4

Duplicates removed (%)

29.7

Number of peaks

20455 (qval < 1E-05)