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Sample GSM4119258

Query DataSets for GSM4119258

Status

Public on Jul 29, 2020

Title

plc-mu atac-seq

Sample type

SRA

Source name

Hepatocellular carcinoma cells

Organism

Homo sapiens

Characteristics

genotype: heterozygote for rs290487 C/C genotype.

Growth protocol

The PLC-PRF-5 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM supplemented with 10% fetal bovine serum.

Extracted molecule

genomic DNA

Extraction protocol

RNA-Seq:Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Double-stranded complementary DNAs were synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (Invitrogen). The cDNAs were then fragmented.
ATAC-Seq: 50,000 cells for each sample were pelleted by centrifugation and lysed in lysis buffer containing 0.1% NP40, 0.1% Tween 20 and 0.01% Digitonin to obtain nucleic. The nucleic pellets were treated with TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) according to manufacturer’s protocols. Immediately following transposition, DNA fragments were purified with MinElute PCR Purification Kit (QIAGEN) and PCR amplified with primers including barcode for a total of 10-12 cycles. The resulting libraries were purified and assessed with Qubit 3 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent).
CHIP-Seq: cells were harvested and fixed. Chromatin was isolated by adding Lysis Buffer (Active Motif), followed by homogenization with Dounce homogenizer. Soluble chromatin was quantified by Nanodrop 2000 (ThermoFisher Scientific). ChIP was performed using Dynabeads Protein G (ThermoFisher Scientific) and 4 μL antibody against TCF4/TCF7L2 (Catalog number 2569, Cell Signaling Technology) with 20-50 μg soluble chromatins. Rabbit IgG (Catalog number ab172730, Abcam) was used for negative control. The beads-antibody-chromatin complexes were washed using ChIP-IT Express Enzymatic (Active Motif) according to manufacturer’s protocols. The chromatin was reverse cross linked overnight at 65°C, treated with RNase A and proteinase K, respectively. The DNA was cleaned up with QIAGEN MinElute PCR Purification Kit (QIAGEN).
RNA-Seq:using the Illumina paired-end RNA-seq approach according to the standard protocol.
ATAC-Seq:The libraries were sequenced on Illumina Hiseq XTen instrument (Illumina).Briefly, 50,000 cells for each sample were pelleted by centrifugation and lysed in lysis buffer containing 0.1% NP40, 0.1% Tween 20 and 0.01% Digitonin to obtain nucleic. The nucleic pellets were treated with TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) according to manufacturer’s protocols. Immediately following transposition, DNA fragments were purified with MinElute PCR Purification Kit (QIAGEN) and PCR amplified with primers including barcode for a total of 10-12 cycles. The resulting libraries were purified and assessed with Qubit 3 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent).
CHIP-Seq: ChIP-seq libraries were prepared using KAPA Hyper Prep Kit and sequenced on Illumina Hiseq XTen instrument.Briefly, cells were harvested and fixed. Chromatin was isolated by adding Lysis Buffer (Active Motif), followed by homogenization with Dounce homogenizer. Soluble chromatin was quantified by Nanodrop 2000 (ThermoFisher Scientific). ChIP was performed using Dynabeads Protein G (ThermoFisher Scientific) and 4 μL antibody against TCF4/TCF7L2 (Catalog number 2569, Cell Signaling Technology) with 20-50 μg soluble chromatins. Rabbit IgG (Catalog number ab172730, Abcam) was used for negative control. The beads-antibody-chromatin complexes were washed using ChIP-IT Express Enzymatic (Active Motif) according to manufacturer’s protocols. The chromatin was reverse c ross linked overnight at 65°C, treated with RNase A and proteinase K, respectively. The DNA was cleaned up with QIAGEN MinElute PCR Purification Kit (QIAGEN).

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

HiSeq X Ten

Description

atac-seq.txt

Data processing

RNA-Seq:index of the reference genome was built and paired-end clean reads were aligned to the reference genome using the software package TopHat2 (v2.0.9) with Bowtie2 (v2.3.4.1). HTSeq (v0.6.1) was used to count the reads numbers mapped to each gene. DESeq2 R package (1.10.1) was used to performe differential expression analysis. Enrichment analysis of differentially expressed genes was performed by KOBAS 3.0 analysis (http://kobas.cbi.pku.edu.cn/index.php).
ATAC-Seq: The resulting libraries were purified and assessed with Qubit 3 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent).
ChIP-Seq:ChIP was performed using Dynabeads Protein G (ThermoFisher Scientific)
Genome_build: mm9
Supplementary_files_format_and_content: SPSS version 13.0 (SPSS Inc., Chicago, IL) was used to complete the statistical analysis. A P value of < 0.05 was considered statistically significant. Quantitative variables were described as the mean ± SD or median (interquartile range) and compared using student t test or Mann-Whitney test. Categorical variables were presented as values (percentages) and compared using Pearson’s χ2 test. Correlation analysis was performed by Pearson linear regression.

Submission date

Oct 11, 2019

Last update date

Jul 29, 2020

Contact name

ZHANG XUEYOU

E-mail(s)

zxy2012@zju.edu.cn

Phone

18806817340

Organization name

Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, China