GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3479297

Query DataSets for GSM3479297

Status

Public on Oct 28, 2020

Title

IRF6.FL.6.0.E6.cycle4

Sample type

SRA

Source name

Synthesized DNA

Organism

synthetic construct

Characteristics

library strategy: SELEX
tf: IRF6
tf type: FL (Full Length)
cycle: 4
plate: 2_E6
molecule subtype: Synthesized DNA

Treatment protocol

Fugene HD (Promega, E2311) was used for plasmid transfection. Specifically, 2 µg of STARR-seq plasmids were mixed with 5 µl of transfection reagents for transfection into 300,000 cells cultured in a single well of 6-well plate. For siRNA transfection, HiPerfect transfection was used following the manufacture guidance. For each experiment, 50 nM of siRNA was used with 5 ul of HiPerfect reagent to make the transfection complex for 1-3×104 cells. Cells were continued to be cultured for 72 hours. The siRNAs targeting human HLF (cat. #GS3131) and MAFG (cat. #GS4097) were commercially available from Qiagen. Silencer negative control siRNA was commercially manufactured and order from Thermo Fisher (cat. #AM4635).

Growth protocol

The HEK293T cells (ATCC, CRL-3216) and HepG2 (ATCC, HB-8065) cells were cultured under normal condition with 5% CO2 at 37°C.

Extracted molecule

genomic DNA

Extraction protocol

ChIP-seq: Briefly, the cells were crossed linked with 1% formaldehyde at ambient temperature for 10 min. The reaction was quenched by 125mM glycine for 5 min at room temperature. Cells were washed with PBS and treated with hypotonic buffer (20mM Hepes pH7.9, 10mM KCl, 1mM EDTA, 10% Glycerol and 1mM DTT with additional protease inhibitor (Roche)) to isolate nuclei. The nuclei were suspended with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate with protease inhibitor) and sonicated using Covaris S220 Focused-ultrasonicator. Fragmented chromatin was pre-cleared with protein G conjugated sepharose beads (GE). Antibodies were used to pull down the respective proteins and their associated chromatin. Washes with different concentration of NaCl were performed. The enriched protein-DNA complexes were reverse crosslinked at 65°C over night with proteinase K (NEB). DNA was purified with Qiagen MinElute kit. STARR-seq: The plasmid pool was transfected into HEK293T or HepG2 cell lines using Fugene HD and continued culturing for 48 hours before harvest. Total RNA was extracted with RNeasy kit (Qiagen, 74104) and mRNA was enriched with poly(dT)25 Dynabeads (Invitrogen, 61002). First strand cDNA was synthesized using a specific primer (5’-CAAACTCATCAATGTATCTTATCATG) with high High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, 4368814). The nested PCR was used to amplify the SNP specific fragments from cDNA, first using two reporter-specific PCR primers (5’-GGGCCAGCTGTTGGGGTGTCCAC & 5’-CTTATCATGTCTGCTCGAAGC) and then generic primers used in HT-SELEX. DNA was purified with AMPure beads. Hi-C: Briefly, the human islets were washed with cold PBS and cut into small pieces. For HepG2 cells, the cells were trypsinized and washed with PBS. The chromatin was cross-linked with 1% formaldehyde (Sigma) at ambient temperature for 10 min and quenched with 125mM glycine for 5 min. PBS washed tissue was homogenized with loose fitting douncer for 30 strokes before centrifugation to isolate the nuclei. Nuclei were isolated and directly applied for digestion using 4 cutter restriction enzyme MboI (NEB) at 37 °C o/n. The single strand overhang was filled with biotinylated-14-ATP (Life Tech.) using Klenow DNA polymerase (NEB). Different from tradition Hi-C, with in situ protocol the ligation was performed when the nuclear membrane was still intact. DNA was ligated for 4h at 16 °C using T4 ligase (NEB). Protein was degraded by proteinase K (NEB) treatment at 55 °C for 30 min. The crosslinking was reversed with 500 mM of NaCl and heated at 68 °C o/n. DNA was purified and sonicated to 300-700 bp small fragments. Biotinylated DNA was selected with Dynabeads My One T1 Streptavidin beads (Life Tech.). Sequencing library was prepared on beads and intensive wash was performed between different reactions. Whole genome sequencing: The genomic DNA was extracted using Qiagen kit (cat. no. 69506). The DNA was then fragmented with Covaris S220 ultrasonicator to 300-500 bp long. RNA-seq: The total RNA was isolated using Qiagen RNeasy mini kit. The sequencing library was prepared using Truseq illumine RNA Library Prep Kit v2 (cat. #RS-122-2001). SELEX: Briefly, the E. coli expressed 6xHis-tagged TF proteins were immobilized to Ni sepharose beads (GE, 17-5318-01) in Promega binding buffer (10mM Tris pH7.5, 50mM NaCl, 1mM MgCl2, 4% glycerol, 0.5mM EDTA, 5µg/ml poly-dIdC). Oligos from input or previous HT-SELEX cycles were added into the protein beads mixture and incubated at ambient temperature for 30 min. After binding, the beads were consecutively washed for 12 times with the Promega binding buffer. After final wash, TE (10mM Tris pH 8.0, 1mM EDTA) was used to re-suspend the beads and for PCR amplification. The PCR products from each HT-SELEX cycle were purified (Qiagen, 28004).
ChIP-seq:
Briefly, the cells were crossed linked with 1% formaldehyde at ambient temperature for 10 min. The reaction was quenched by 125mM glycine for 5 min at room temperature. Cells were washed with PBS and treated with hypotonic buffer (20mM Hepes pH7.9, 10mM KCl, 1mM EDTA, 10% Glycerol and 1mM DTT with additional protease inhibitor (Roche)) to isolate nuclei. The nuclei were suspended with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate with protease inhibitor) and sonicated using Covaris S220 Focused-ultrasonicator. Fragmented chromatin was pre-cleared with protein G conjugated sepharose beads (GE). Antibodies were used to pull down the respective proteins and their associated chromatin. Washes with different concentration of NaCl were performed. The enriched protein-DNA complexes were reverse crosslinked at 65°C over night with proteinase K (NEB). DNA was purified with Qiagen MinElute kit. STARR-seq:
The plasmid pool was transfected into HEK293T or HepG2 cell lines using Fugene HD and continued culturing for 48 hours before harvest. Total RNA was extracted with RNeasy kit (Qiagen, 74104) and mRNA was enriched with poly(dT)25 Dynabeads (Invitrogen, 61002). First strand cDNA was synthesized using a specific primer (5’-CAAACTCATCAATGTATCTTATCATG) with high High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, 4368814). The nested PCR was used to amplify the SNP specific fragments from cDNA, first using two reporter-specific PCR primers (5’-GGGCCAGCTGTTGGGGTGTCCAC & 5’-CTTATCATGTCTGCTCGAAGC) and then generic primers used in HT-SELEX. DNA was purified with AMPure beads.
Hi-C:
Briefly, the human islets were washed with cold PBS and cut into small pieces. For HepG2 cells, the cells were trypsinized and washed with PBS. The chromatin was cross-linked with 1% formaldehyde (Sigma) at ambient temperature for 10 min and quenched with 125mM glycine for 5 min. PBS washed tissue was homogenized with loose fitting douncer for 30 strokes before centrifugation to isolate the nuclei. Nuclei were isolated and directly applied for digestion using 4 cutter restriction enzyme MboI (NEB) at 37 °C o/n. The single strand overhang was filled with biotinylated-14-ATP (Life Tech.) using Klenow DNA polymerase (NEB). Different from tradition Hi-C, with in situ protocol the ligation was performed when the nuclear membrane was still intact. DNA was ligated for 4h at 16 °C using T4 ligase (NEB). Protein was degraded by proteinase K (NEB) treatment at 55 °C for 30 min. The crosslinking was reversed with 500 mM of NaCl and heated at 68 °C o/n. DNA was purified and sonicated to 300-700 bp small fragments. Biotinylated DNA was selected with Dynabeads My One T1 Streptavidin beads (Life Tech.). Sequencing library was prepared on beads and intensive wash was performed between different reactions.
Whole genome sequencing:
The genomic DNA was extracted using Qiagen kit (cat. no. 69506). The DNA was then fragmented with Covaris S220 ultrasonicator to 300-500 bp long.
RNA-seq:
The total RNA was isolated using Qiagen RNeasy mini kit. The sequencing library was prepared using Truseq illumine RNA Library Prep Kit v2 (cat. #RS-122-2001).
SELEX:
Briefly, the E. coli expressed 6xHis-tagged TF proteins were immobilized to Ni sepharose beads (GE, 17-5318-01) in Promega binding buffer (10mM Tris pH7.5, 50mM NaCl, 1mM MgCl2, 4% glycerol, 0.5mM EDTA, 5µg/ml poly-dIdC). Oligos from input or previous HT-SELEX cycles were added into the protein beads mixture and incubated at ambient temperature for 30 min. After binding, the beads were consecutively washed for 12 times with the Promega binding buffer. After final wash, TE (10mM Tris pH 8.0, 1mM EDTA) was used to re-suspend the beads and for PCR amplification. The PCR products from each HT-SELEX cycle were purified (Qiagen, 28004).

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

processed data file:
pbs.obs_pval05.tsv
IRF6_FL_cycle4_Protein6_E6_S1302_L007

Data processing

ChIP-seq: Reads were aligned using BWA MEM with either single-end or pair-end model to the hg19 reference genome. Reads with low mapping quality (mapq<10) were filtered out, and PCR duplicates were removed using Picard tool. MACS2 were then applied to call peaks and generate signals to view in the genome browser.
STARR-seq:STARR-seq reads were aligned to the oligo libraries using BWA with default parameters. Read counts for each oligos were then counted using custom scripts. Counts for technical replicates were merged. Oligos were filtered to keep only oligos covered by more than 25 reads in the input library and more than five reads in at least three libraries.
Hi-C:Briefly, each end of read pairs were aligned separately using BWA MEM to the hg19 reference genome with default parameters. Chimeric read ends were further processed to keep only the five-prime alignment. Read ends with low mapping quality (mapq<10) were removed, and remaining read ends were paired using custom scripts. PCR duplicates were removed using Picard tool (http://broadinstitute.github.io/picard). Aligned reads were further transformed to the juicer format and processed into hic format using juicebox tool. Chromatin loops were called using Hiccups with default parameters.
WGS:Reads from whole genome sequencing (WGS) were aligned using BWA MEM in pair-end model with default parameters. PCR duplicates were removed using Picard tools (http://broadinstitute.github.io/picard). Variants were then called according to the GATK best practice pipeline using GATK 3.6-0.
RNA-seq:Reads were aligned to the hg19 reference genome using STAR 2.4.2a with default parameters in pair-end model. Only uniquely aligned reads were kept for further analysis. Cufflinks 2.2.1 was used to compute FPKM for each gene.
SELEX:Sequencing data of each HT-SELEX cycle was aligned to the oligo library using BWA. More specific details to identify pbSNPs can be found in methods of associated manuscript.
Genome_build: hg19
Supplementary_files_format_and_content:
ChIP-seq: Peak files in narrowPeak format and signal files in bigWig format for each samples were provided.
STARR-seq:Counts for each STARR-seq experiments were provided.
Hi-C:Juicebox hic format contains contact matrices in multiple resolutions and list of loops in text format were provided.
WGS:VCF file for all variants and phased variants in HepG2 cells were provided.
RNA-seq:Text files including FPKM values for all genes in each sample were provided.
SELEX:Text file summarizing measurements of allelic TF binding to all SNPs tested was included.

Submission date

Nov 19, 2018

Last update date

Nov 01, 2020

Contact name

Yunjiang Qiu

Organization name

University of California, San Diego

Street address

9500 Gilman Drive

City

La Jolla