GSM4043681: xChIP H1.5 rep3; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H1.5
Cell type
Cell type Class
Blood
Cell type
HL-60
Primary Tissue
Blood
Tissue Diagnosis
Leukemia
Attributes by original data submitter
Sample
source_name
HL-60/S4
cell line
HL-60/S4
antibody
H1.5
experiment
xChIP, no treatment
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For both single-fixation and double-fixation ChIP (xChIP and xxChIP, see Fig. 1), chromatin was disrupted with a Covaris Focused Ultrasonicator M220. In xChIP, each frozen cell pellet (1 cryovial) was dispersed in 130 µl of Covaris Sonication Buffer (1 mM EDTA, 10 mM Tris [pH 7.6], 0.1 % SDS), followed by sonication (20 min, 200 cycles, 75 Watts, Duty Cycle 20%, 7oC). The sonicates were centrifuged at 18,000xg, 10 min, 40C and the supernatants recovered. SDS was reduced in the supernatants to ~0.003% and replaced with 0.05% Tween-20, employing repeated dilution with PBST (PBS+0.05% Tween-20) and centrifugal concentration using a Centricon YM-50. Six centrifugations of ~1/2 dilutions with PBST at 1000xg, 10 min resulted in ~0.5 ml of the final retentate with reduced SDS. IgG-free BSA (Sigma A3294) was added to a final BSA concentration of 5%. All ChIP-Seq experiments were performed on undifferentiated HL-60/S4 cells that were fixed, permeabilized and stored in cryovials (containing ~107 cells/cryovial) in liquid Nitrogen. Cells had been harvested from growth medium at ~106 cells/ml, centrifuged and washed with PBS, fixed in 1% HCHO/PBS for 10 min at RT, stopped with 0.125 M glycine for 5 min, washed with PBS, followed by PBS + 0.1 M PMSF. The fixed cells were permeabilized for 10 min at 4oC in a lysis buffer containing 25 mM HEPES buffer (pH 7.8), 1 mM MgCl2, 10mM KCl, 0.1% NP40, 1 mM DTT and 0.5 mM PMSF. Following centrifugation and removal of supernatants, cell pellets were frozen in residual lysis buffer at liquid Nitrogen temperature. For both single-fixation and double-fixation ChIP (xChIP and xxChIP, see Fig. 1), chromatin was disrupted with a Covaris Focused Ultrasonicator M220. In xChIP, each frozen cell pellet (1 cryovial) was dispersed in 130 µl of Covaris Sonication Buffer (1 mM EDTA, 10 mM Tris [pH 7.6], 0.1 % SDS), followed by sonication (20 min, 200 cycles, 75 Watts, Duty Cycle 20%, 7oC). The sonicates were centrifuged at 18,000xg, 10 min, 40C and the supernatants recovered. SDS was reduced in the supernatants to ~0.003% and replaced with 0.05% Tween-20, employing repeated dilution with PBST (PBS+0.05% Tween-20) and centrifugal concentration using a Centricon YM-50. Six centrifugations of ~1/2 dilutions with PBST at 1000xg, 10 min resulted in ~0.5 ml of the final retentate with reduced SDS. IgG-free BSA (Sigma A3294) was added to a final BSA concentration of 5%. In xxChIP, the once-fixed frozen cell pellets were dispersed in a buffer reminiscent of the permeabilizing buffer used in immunostaining reactions (0.1% Triton X-100, 0.1 mM PMSF plus Sigma Protease Inhibitor Cocktail [P8340]) for 20 min at RT. After PBS washes, the permeabilized cells were suspended in PBST+5% IgG-free BSA (PBSTB) and rotated for 90 min at RT. To 300 µl aliquots containing ~6x107 cells, the primary antibody was added: 30 µl anti-histone H1.2 (1 mg/ml) or 60 µl anti-histone H1.5 (0.5 mg/ml). The cells plus antibody were rotated for 4 hours at RT. Following antibody incubation, the cells were washed several times with PBS to remove unbound antibody. They were made 1% HCHO/PBS for the second fixation and rotated 2.5 minutes at RT. Fixation was stopped with 0.125 M glycine for 5 min, cells washed with PBS and dispersed in 1.0 ml of Covaris Sonication Buffer (1 mM EDTA, 10 mM Tris [pH 7.6], 0.1 % SDS), followed by sonication at optimized conditions (40 min, 400 cycles, 75 Watts, Duty Cycle 26%, 7oC). The sonication buffer was replaced with PBST, employing centrifugal concentration, as described above. Library preparation was performed by the DKFZ sequencing facility following a standard Illumina protocol Illumina NEBNext Ultra