 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 17, 2024 |
| Title |
BRC patient P2, normal breast tissue, MNase-seq |
| Sample type |
SRA |
| |
|
| Source name |
breast
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: breast
cell type: breast
condition: normal
|
| Treatment protocol |
n/a
|
| Growth protocol |
n/a
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MNase-seq and MNase-assisted histone H3 ChIP-seq Chromatin extraction. Paired normal and tumour breast tissues extracted during surgery were stored at -80 ͦC. Frozen tissue was first ground into fine powder in liquid nitrogen. It was further homogenized by manual douncing in 5 ml ice-cold Buffer I (0.3 M Sucrose in 60 mM KCl, 15 mM NaCl, 5mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl (pH 7.5) with freshly added 0.5 mM DTT, 0.1 mM PMSF and 1XProtease Inhibitor Cocktail, Thermo Scientific Halt Protease Inhibitor Single-use Cocktail (100X). The homogenized cell suspension was centrifuged in a 15 ml Falcon conical centrifuge tube at 6000 g for 10 minutes at 4 ͦC. The precipitated cells were re-suspended in 500 µL of Buffer I, then 500 µL of Buffer II (same as Buffer I, but with freshly added 0.4 % (v/v) IGEPAL CA-630) was gently added on top and the lysate was incubated on ice for 3 minutes, followed by centrifugation at 3000 g for 3 minutes at 4 ͦC. The pellet was re-suspended in 1 ml of ice-cold NBR Buffer (85 mM NaCl, 5.5 % Sucrose, 10 mM Tris-HCl pH 7.5, 3 mM MgCl2, 1.5 mM CaCl2 with freshly added 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM DTT) and centrifuged at 3000 g for 3 minutes at 4 ͦC. The pellet then was re-suspended in 200 µl of NBR buffer. Samples were treated with 1 µl RNAseA (10 mg/ml) per 100 µl of nuclear lysate for 5 minutes at room temperature. MNase digestion. MNase restriction enzyme (Sigma, Cat No: N5386-500UN) was diluted to 1U (Sigma Boehringer Units) in MNase buffer (50 % Glycerol, 10 mM Tris p H7.6 and 50 mM NaCl). The pellets re-suspended in 200 µl of NBR buffer in the chromatin extraction step were digested with MNase enzyme as follows. Chromatin derived from paired normal and tumour tissue was diluted with NBR buffer to keep the same concentration in both preparations. A 20 µl aliquot of purified nuclei was test digested with 1 µl MNase for 7 min at room temperature to assess the optimal digestion time and enzyme concentrations. 1 µg of chromatin was digested with 1U of MNase for 7 minutes at room temperature to achieve 80:20 relative abundance of mono- and di-nucleosome fractions on agarose gel. The MNase digestion was stopped by addition of equal volume (NBR:STOP, 1:1) of STOP Buffer (215 mM NaCl, 10 mM TrisHCl pH 8, 20 mM EDTA, 5.5 % Sucrose, 2 % TritonX 100 with freshly added 0.2 mM PMSF, 1 mM DTT and 2X Protease Inhibitors). After digestion, chromatin was incubated on ice in the fridge at 4 ͦC overnight to release the digested chromatin. Chromatin Immunoprecipitation (ChIP). 10 µl of Protein A beads (DynabeadsTM Protein A; Invitrogen) equivalent to 10 µl bed volume of slurry were prepared per each ChIP reaction. Bead storage buffer was aspirated, and the beads were washed twice in 500 µl in Block solution (0.5 % BSA (w/v) in PBS supplemented with 0.1 mM PMSF). Beads sufficient for 4 ChIP samples were all combined and dissolved in 600 µl of Block solution with addition of 5 µg of anti-H3 antibody Abcam AB1791 per ChIP reaction (20 µg for 40 µl of beads for 4 ChIP samples). The antibody-bead solution was incubated on a slow rotating wheel at 4 ͦC for two hours. After incubation, the beads were again washed in 600 µl block solution to remove un-bound antibody. The chromatin incubated overnight after MNase digestion, was then centrifuged for 10 minutes at max speed 9800 g at 4 ͦC. The supernatant with released chromatin was collected and stored at -20 ͦC and used for ChIP experiment. 5 % of chromatin volume used for MNase-assisted ChIP-seq was frozen at -20 ͦC as Input and used for MNase-seq. 300 µl of chromatin was incubated with the antibody bound Protein A beads for 3 hours on a rotating wheel at 4 ͦC. Unbound chromatin was washed 4 times in 1 ml volume of ChIP-Wash buffer (150 mM NaCl, 10 mM Tris-HCl pH 8, 2 mM EDTA, 1 % NP40, 1 % Nadeoxycholate with freshly added 0.5 mM DTT, 0.1 mM PMSF and 1XProtease Inhibitor Cocktail). The initial wash of bound beads was done by gently mixing beads in the ChIP-Wash buffer for one min. For subsequent 3 washes: the tube was rotated for 5 minutes at 4 ͦC on a rotating wheel, then placed in magnetic rack for 1 min at 4 ͦC and then the supernatant was withdrawn. After two washes the beads with wash buffer were transferred to new tubes. And as a final step, the beads were washed with 1 ml of filtered 1x TE buffer (10 mM Tris-HCl containing 1 mM EDTA) at room temperature, after which the TE buffer was removed as above. The elution of bound chromatin was performed with 100 µl of Elution buffer (0.1 M NaHCO3, 1 % SDS) supplemented with 2 µl of Proteinase K per ChIP sample tube. The beads were incubated with the elution buffer for 6-12 hours at 55 ͦC. Similarly, 2 µl of Proteinase K were added to Input material and incubated at 55 ͦC for 6-12 hours. The incubated beads were briefly vortexed and supernatant collected by placing the tubes in a magnetic rack. The beads were additionally washed in 20 µl of elution buffer and supernatant collected as above. The eluants were further purified with PCR purification columns according to manufacturer’s instructions and eluted in 20 µl volume of elution buffer of the kit. These final eluants were stored at -20 ͦC ready for library preparation step. cfDNA extraction. Cell-free DNA was extracted from 500 µl of blood plasma or serum as detailed below, following the instructions of the purification kits. For patient P1, cfDNA was extracted from 500 µl of blood serum using Plasma/serum Cell-Free Circulating DNA purification Mini Kit (Norgen Biotek Corp). For patient P2, cfDNA was extracted from 500 µl
The libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, NEB #E7103) according to the manufacturer’s instructions.
NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, NEB #E7103)
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Breast tissue samples for patients P1-P3 were sequenced on Illumina NovaSeq S4, TruSeq ChIP Library Prep Kit (IP-202-1012), 2 x 100 bp paired-end mode. cfDNA samples for patients P1 and P3 were sequenced as above, but in 2 x 150 bp paired-end mode. Samples for patient P4 were sequenced on Illumina HiSeq 2500 sequencer, 2 x 75 bp paired-end mode, using NEBNext Ultra II Kit from NEB for library preparation. Paired-end sequenced reads were mapped with Bowtie to human genome hg19, considering only uniquely mapped reads and allowing up to one mismatch. This resulted in ~500 million mapped reads per sample (see details in Supplementary Table S1). NucTools pipeline was used to obtain BED files with mapped nucleosomal DNA fragments including information about DNA fragment sizes. A set of Perl scripts called cfDNAtools developed for the next steps of analysis reported here is available at https://github.com/TeifLab/cfDNAtools. cfDNAtools script extract_nuc_sizes.pl was used to extract fractions with DNA fragments with sizes between 100-120 bp, 120-140 bp, 140-160 bp, 160-180 bp and 180 -200 bp. Intersections between regions of interest were done using BEDTools (Quinlan, 2014).
DNA fragments with sizes 120-180bp are included in this dataset
Assembly: hg19
Supplementary files format and content: BED files containing all mapped reads within size range 120-180bp. The FED file contains the following columns: chromosome, start coordinate, end coordinate, fragment size (bp)
Supplementary files format and content: BigWig files for visualisation in the genome browser
|
| |
|
| Submission date |
Aug 01, 2022 |
| Last update date |
Apr 17, 2024 |
| Contact name |
Vladimir B Teif |
| E-mail(s) |
vteif@essex.ac.uk
|
| Organization name |
University of Essex
|
| Department |
School of Life Sciences
|
| Street address |
Wivenhoe Park
|
| City |
Colchester |
| ZIP/Postal code |
CO4 3SQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE210250 |
Nucleosome reorganisation in breast cancer tissues |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6424690_BRC-P2-N-MNase-120-180.bed.gz |
4.4 Gb |
(ftp)(http) |
BED |
| GSM6424690_BRC-P2-N-MNase-120-180.bw |
1.9 Gb |
(ftp)(http) |
BW |
| GSM6424690_P2_N_MNase_resequenced_120-180.bed.gz |
6.8 Gb |
(ftp)(http) |
BED |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
|
|
|
|
 |
