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| Status |
Public on Jun 14, 2020 |
| Title |
xChIP H1.5 rep1 |
| Sample type |
SRA |
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| Source name |
HL-60/S4
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HL-60/S4
antibody: H1.5
experiment: xChIP, no treatment
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| Growth protocol |
The human myeloid leukemic cell line HL-60/S4 (ATCC, CRL-3306) was maintained in RPMI-1640 medium plus 10% FCS and 1% Pen/Strep/Glut. Cells were grown in 6 ml of medium in T-25 flasks, generally split (1:6-1:12) 2-3 times/week. For large scale preps, cells were grown in T-75 flasks with up to 30 ml media. Cell concentrations were monitored using a hemocytometer. Rabbit polyclonal ChIP-Grade antibodies were obtained from Abcam: anti-histone H1.2 (ab4086) and anti-H1.5 (ab18208). Both antibodies are directed against antigenic determinants within the N-terminal 1-100 aa residues.
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| Extracted molecule |
genomic DNA |
| Extraction 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
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
xChIP H1.5 rep1
XChIP_H1.5.bed.tdf
XChIP_H1.5.bed
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| Data processing |
Genome_build: hg19
BED files with MUSIC-defined peaks
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| Submission date |
Aug 23, 2019 |
| Last update date |
Jun 14, 2020 |
| Contact name |
Vladimir B Teif |
| E-mail(s) |
[email protected]
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| Organization name |
University of Essex
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| Department |
School of Life Sciences
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| Street address |
Wivenhoe Park
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| City |
Colchester |
| ZIP/Postal code |
CO4 3SQ |
| Country |
United Kingdom |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE136264 |
Histone H1 distribution and epitope exposure within chromatin high-order structure |
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| Relations |
| BioSample |
SAMN12628184 |
| SRA |
SRX6765532 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4043679_ChIPH_1_dot_5_11-11-14.bw |
134.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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