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Sample GSM2627219

Query DataSets for GSM2627219

Status

Public on Nov 01, 2017

Title

RWPE1_HiC_rep1

Sample type

SRA

Source name

RWPE1

Organism

Homo sapiens

Characteristics

cell line: prostate cell line RWPE1
treatment: None
genotype/variation: wild type

Treatment protocol

For overexpression, RWPE-1 cells were transfected with equal amounts of a HOXA13 expression vector (GeneCopoeiaTM cat. no. EX-I0919-M14) or the corresponding control plasmid expressing eGFP (GeneCopoeiaTM cat. no. EX-EGFP-M14) and harvested 48 hours later. Cells were analyzed by FACS to identify GFP-positive cells and the top 10% eGFP positive cells from 3 control and 3 HOXA13-expressing flasks were collected by cell sorting and subjected to RNA isolation, followed by RNA-seq analysis.

Growth protocol

RWPE-1 cells were cultured in Keratinocyte Serum Free Medium (Thermo Fisher Scientific, cat. no. 17005-042) without antibiotics. To passage the cells, 0.05% trypsin-0.02% EDTA solution was used and the trypsinization was stopped by adding Dulbecco's phosphate-buffered saline containing 2% FBS. Cells were grown at 37 °C with 5% CO2 for under 20 passages. The cell line identity was confirmed using the STR cell line authentication method at the Norris Comprehensive Cancer Center Media Core and was shown to be free of mycoplasma using the universal mycoplasma detection kit (ATCC 30-1012K).

Extracted molecule

genomic DNA

Extraction protocol

RNA was prepared using TRIzol reagent (Thermal Fisher Scientific, cat. no. 15596018). In situ Hi-C experiments were performed in RWPE-1 cells following the original protocol by Rao et al with minor modifications. Hi-C was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. Hi-C material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). Hi-C library was amplified with 14 cycles of PCR using Illumina primers. Each library was sequenced with 75bp paired-end for ~500M read pairs using Illumina HiSeq 2000.
The RNA-seq libraries were made using KAPA stranded mRNA-seq kits (KAPK Biosystems, cat. no. kk8421) according to manufacturer’s instructions
In situ HiC experiments were performed in RWPE-1 cells following the original protocol by Rao et al with minor modifications. HiC was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. HiC material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). HiC library was amplified with 14 cycles of PCR using Illumina primers.

Library strategy

Hi-C

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

RWPE1_comb_HiC_5k.raw.matrix.txt
RWPE1_comb_HiC_40k.raw.matrix.txt

Data processing

RNA-seq data was inspected by FastQC and trimmed using Trimmomatic version 0.35. Reads were mapped to human genome GRCh37 using STAR version 2.5.2b with default settings.
Alignment results were processed for gene quantification using featureCounts version 1.5.1 with the strand-specific option
Differentially expressed genes were determined using DESeq2 version 1.14.1
TPM values for each gene were calculated by RSEM v1.3.0
The quality of each Hi-C library was checked with FastQC and HICUP version 0.5.842. Raw fastq files were processed through the HiC-Pro version 2.8.0 to make the raw contact count matrices for multiple resolutions (5kb, 10kb, 20kb, 40kb, 100kb). The matrices were normalized using the iterative correction method (iced python library). For the identification of topologically associating domains (TADs), the normalized Hi-C contact matrices, binned with 40kb resolution were used and the domains were called using the Domain caller. Intra-chromosomal significant loops (50kb-10Mb range) were selected with the 5kb resolution contact count matrices using the Fit-Hi-C (false discovery rate (FDR) < 0.05). Hi-C chromatin interaction heatmaps with 5kb resolution was visualized in the 3D Genome Browser (http://biorxiv.org/content/early/2017/02/27/112268). To increase the sequencing depth, two replicates were pooled and processed as above described.
Genome_build: GRCh37
Supplementary_files_format_and_content: TPM values and differential expression; Hi-C contact matrices

Submission date

May 15, 2017

Last update date

May 15, 2019

Contact name

Zhifei Luo

E-mail(s)

Zhifeilu@usc.edu

Organization name

University of Southern California

Street address

1450 Biggy Street, NRT 6514