 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 12, 2022 |
| Title |
LNCaP vehicle |
| Sample type |
SRA |
| |
|
| Source name |
Prostate
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Prostate cancer cell line
treatment: DHT starved
|
| Treatment protocol |
Cross Linking: 10 million cells were washed three times in chilled 1X phosphate buffered saline (PBS) in a 50 mL centrifuge tube, and pelleted by centrifugation at 500 xg for 5 min. at 4°C between each wash. Cells were re-suspended in 10 mL room temperature 1X PBS 1% formaladehyde [EMD Millipore cat no. 818708] by gently pipetting with a wide-bore tip, then incubated at room temperature for 10 min. To quench the cross-linking reaction, 527 μL of 2.5 M glycine was added to achieve a final concentration of 1% w/v or 125 mM in 10.5 mL. Cells were incubated for 5 min. at room temperature followed by 10 min. on ice. The cross-linked cells were pelleted by centrifugation at 500 xg for 5 min. at 4°C. Restriction enzyme digest: Each cell pellet was resuspended in a mixture of 50 μL of protease inhibitor cocktail [Sigma Aldrich cat no. P8340] in 500 μL of cold permeabilization buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630) and placed on ice for 15 min. Cells were centrifuged at 500 xg for 10 min. at 4°C after which the supernatant was aspirated and replaced with 200 μL of chilled 1.5X digestion reaction buffer [NEB ] compatible with the restriction enzyme used. Cells were centrifuged again at 500 xg for 10 min. at 4°C, then aspirated and re-suspended in 300 μL of chilled 1.5X digestion reaction buffer. To denature the chromatin, 33.5 μL of 1% w/v SDS [Thermo Fisher Scientific cat no. 15553027] was added to each cell suspension and incubated for exactly 10 min. at 65°C with gentle agitation, and then placed on ice immediately afterwards. To quench the SDS, 37.5 μL of 10% v/v Triton X-100 [Sigma Aldrich cat no. 93443] was added for a final concentration of 1%, followed by incubation for 10 min. on ice. Permeablized cells were then digested with a final concentration of 1 U/μL of either DpnII, NlaIII or HindIII [NEB] brought to volume with nuclease-free water to achieve a final 1X digestion reaction buffer in 450 μL. Cells were then mixed by gentle inversion. Cell suspensions were incubated in a thermomixer at 37°C for 18 hours with periodic < 1000 rpm rotation (< 30 sec every 15 min.) to prevent condensation inside the lid. Proximity ligation and reverse cross-linking: DpnII and NlaIII restriction digests were heat inactivated at 65°C for 20 min. with 300 rpm rotation. HindIII digests were chemically inactivated with a final concentration of 0.1% w/v SDS at 65°C for 20 min. with 300 rpm rotation, then quenched with a final concentration of 1% v/v Triton X-100 [Sigma Aldrich cat no. 93443].Proximity ligation was set up at room temperature with the addition of the following reagents: 100 μL of 10X T4 DNA ligase buffer [NEB], 10 μL of 10 mg/mL BSA and 50 μL of T4 Ligase [NEB M0202L] in a total volume of 1000 μL with nuclease-free water. The ligation was cooled to 16°C and incubated for 6 hours with gentle rotation.
|
| Growth protocol |
LNCaP cells were purchased from the American Type Culture Collection [ATCC CRL-1740), grown on poly-L-lysine coated plates in 5\% Fetal Bovine Serum (FBS) containing RPMI-1640, and incubated at 37C and 5\% CO2. Cells were passaged twice weekly or once cultures reached 80\% confluency. LNCaP cells were starved of hormone for 72 hours then stimulated with vehicle or 1nM DHT for the indicated time points (0 or 18 hours) before fixing with 1\% formaldehyde.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
To reverse cross-link, samples were treated with 100 μL 20 mg/mL Proteinase K [Thermo Fisher Scientific cat no. 25530049], 100 μL 10% SDS [Thermo Fisher Scientific cat no. 15553027] and 500 μL 20% v/v Tween-20 [Sigma Aldrich cat no. P9416] in a total volume of 2000 μL with nuclease-free water. Samples were incubated in a thermomixer at 56°C for 18 hours with < 1000 rpm rotation (< 30 sec every 15 min) to prevent condensation inside the lid. In order to purify DNA, the sample was transferred to a 5 mL centrifuge tube. The original tube was rinsed with a further 200 μL of nuclease-free H2O to collect any residual sample, bringing the total sample volume to 2.2 mL. DNA was then purified from the sample using a standard phenol chloroform extraction and ethanol precipitation.
Purified DNA was SPRI size selected for fragments >1.5 kb, then prepared for sequencing using Oxford Nanopore Technologies SQK-LSK109, and sequenced on ONT's MinION, GridIONor PromethION platforms.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
PromethION |
| |
|
| Data processing |
Basecalling was done using Guppy base caller version 3.4.0rc3 run in hac mode
Pore-C reads were aligned using bwa bwasw with parameters -b 5 -q 2 -r 1 -T 15 -z 10. Further processing done by custom Pore-C pipeline found here https://github.com/nanoporetech/pore-c
CpG methylation was called using f5c (https://github.com/hasindu2008/f5c), an optimized and gpu-compatible re-implementation of the call-methylation module in Nanopolish (https://github.com/jts/nanopolish}. Per-read calls were aggregated into per-locus calls using f5c meth-freq -s with the default log-likelihood ratio threshold.
Pair wise contacts using pore_c to-matrix in the pore-c pipelines with default settings
mcool files were made using cooler zoomify with default parameters
Genome_build: GRCh38
Supplementary_files_format_and_content: .csv Pore-C multi-way contacts files where each row is a Pore-C monomer and metadata related to them including the concatemer they blong to.
Supplementary_files_format_and_content: .csv Pore-C pair-wise contacts files where each row is a pair-wise contact
Supplementary_files_format_and_content: .mcool Pore-C pair-wise cooler contacts files
Supplementary_files_format_and_content: .csv Pore-C phased methylation file, phased in two haplotypes with aggregated methylation
|
| |
|
| Submission date |
Feb 28, 2022 |
| Last update date |
May 12, 2022 |
| Contact name |
Marcin Imielinski |
| E-mail(s) |
[email protected]
|
| Organization name |
Weill Cornell Medicine
|
| Department |
Pathology and Laboratory Medicine
|
| Lab |
Imielinski Lab
|
| Street address |
413 E 69th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL26167 |
| Series (1) |
| GSE149117 |
Pore-C: Combination of chromatin capture assay and Oxford Nanopore Technology long read sequencing. |
|
| Relations |
| BioSample |
SAMN26315986 |
| SRA |
SRX14320238 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5923734_LNCaP_Set1_0hour_PAG48791_GRCh38_unphased.matrix.mcool |
2.8 Gb |
(ftp)(http) |
MCOOL |
| GSM5923734_LNCaP_Set1_0hour_PAG48791_contacts.csv.gz |
3.4 Gb |
(ftp)(http) |
CSV |
| GSM5923734_LNCaP_Set1_0hour_PAG48791_full_pore_c.csv.gz |
1.9 Gb |
(ftp)(http) |
CSV |
| GSM5923734_LNCaP_Set2_0hour_PAG49193_GRCh38_unphased.matrix.mcool |
3.6 Gb |
(ftp)(http) |
MCOOL |
| GSM5923734_LNCaP_Set2_0hour_PAG49193_contacts.csv.gz |
4.1 Gb |
(ftp)(http) |
CSV |
| GSM5923734_LNCaP_Set2_0hour_PAG49193_full_pore_c.csv.gz |
1.9 Gb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
