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Sample GSM1302276

Query DataSets for GSM1302276

Status

Public on Nov 24, 2014

Title

ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Scrambled, 15 h, target protein - Tcf7l2, rep 2

Sample type

SRA

Source name

H4IIE hepatocytes

Organism

Rattus norvegicus

Characteristics

cell type: hepatocytes
cell_line: H4IIE
treatment: siRNA - Scrambled
antibody: TCF7L2 (Lot 2, clone C48H11, cat # 2569, Cell Signaling Technology, Danvers, MA, USA)

Treatment protocol

Silencing of TCF7L2 mRNA and protein levels was achieved using 100 nM Dharmacon SmartPool siRNA (cat #), introduced via Neon electroporation, as described previously (PMID: 21901280). The experiment was performed in 1-3 replicates with separate electroporations being performed on a different passage of cells on a different date.

Growth protocol

Low passage H4IIE cells were purchased from ATCC and used in the lab up to passage 30. The cells were routinely cultured in low glucose (5 mM) DMEM media supplemented with 10% fetal bovine serum in the absence of antibiotics and laboratory stocks tested negative for mycoplasma. After siRNA treatment, cells were immediately plated in 24-well plates in regular culture medium.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin was prepared from approximately 25 million siTCF7L2 or scramble electroporated cells by shearing with MNase using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) to fragments ranging between 150 bp and 900 bp. Each aliquot was incubated overnight with either 5 ul of the anti-TCF7L2 antibody or 1 µl of the anti-HNF4α antibody. A sample consisting of 2% of the total input chromatin was removed from each and served as the sequencing control. Immunoprecipitated material was purified as described by the manufacturer (Cell Signaling Technology), except that replicate immunoprecipitations were pooled at the final Qiagen DNA purification step. Each sequenced sample represents a pool of 2 independent immunoprecipitations, each consisting of between 10 and 20 ug of chromatin.
ChIP-Seq libraries were generated using a combination of Illumina TruSeq adapters/indexes and a Kapa Library Preparation Kit (Kapa Biosystems, Woburn, MA, USA; cat # KK8200) with the following modifications. End-repair was performed on approximately 5 – 10 ng of ChIP sample. Qiagen Minelute purifications (Qiagen, Valencia, CA, USA) were utilized in between each step of adenylation and adapter ligation. Adapter ligation was carried out using stock Illumina adapters and Kapa T4 ligase at 30°C for 10 minutes, followed by a single DNA clean-up using homemade SPRI beads. The adapted material was minimally amplified for 5 cycles, using Illumina PCR primer cocktail and Kapa HiFi HotStart Polymerase ReadyMix (cat # KK2601), to convert y-shaped adapters to double-stranded DNA for more reliable electrophoresis, and then size selected on a 2% agarose gel before a final PCR amplification of 8 – 12 cycles and SPRI bead clean-up. Final libraries were checked for size using Bioanalyzer HS DNA or DNA 1000 chips.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

RNA-seq, transcriptome building: Pooled reads were mapped, using Tophat version 2.0.4 (with underlying Bowtie version 0.12.8), against UCSC known transcripts for rn4, supplemented with additional transcripts from Ensembl, and allowing for the identification of novel exon-exon junctions for the reference genome (rn4 supplemented with EST-based sequence at chr1:262,210,923-262,210,894 to partial fill a gap including a part of an exon of Tcf7l2; corresponding fix was introduced also for the UCSC reference transcriptome). Essential Tophat command arguments were: --library-type fr-unstranded --prefilter-multihits --no-coverage-search -G <UCSC+Ensembl known transcripts>.
RNA-seq, transcriptome building: Transcript mappings were further processed using Cufflinks package version 2.0.2 with essential command arguments, for Cufflinks: --frag-bias-correct --multi-read-correct --upper-quartile-norm -g <UCSC+Ensembl known transcripts> --library-type fr-unstranded, and for Cuffcompare: -r <UCSC+Ensembl known transcripts> -R -d 20 -V <.gff output of Cufflinks>.
RNA-seq, transcriptome building: The initial transcriptome was cleaned by removing all transcripts that were identified as fully silent (FPKM=0), strandless (in Cuffcompare class codes u, o, x, and, if also single-exon, i), or likely artifacts (Cuffcompare class codes e, p, r, s and c). The transcripts that did not represent any known rn4 gene were analyzed against mouse (mm10), human (hg19), as well as an updated rat reference transcriptome sequences using discontiguous megablast with stringent cutoffs to provide official gene symbols for a few additional genes.
RNA-seq, timecourse: Reads for time points 3, 6, 9, 12, 15, 18, 48, and 96 hours after Tcf7l2 or scramble siRNA treatment were mapped against the H4IIE-specific transcriptome using Tophat version 2.0.4 (with underlying Bowtie version 0.12.8). Essential Tophat command arguments were: --library-type fr-unstranded --prefilter-multihits --no-novel-juncs --transcriptome-only -G <H4IIE-specific transcriptome>.
RNA-seq, timecourse: Conversion to sorted BAM format using samtools
RNA-seq, timecourse: Differential expression was analyzed using Cuffdiff version 2.0.2., comparing Tcf7l2 against scramble siRNA treatment. Essential Cuffdiff command arguments were: --FDR 0.05 --library-type fr-unstranded --multi-read-correct --upper-quartile-norm --frag-bias-correct <rn4 genome fastas> <H4IIE-specific transcriptome>
ChIP-seq: Alignment to rn4 using Bowtie v0.12.8 with the following essential command line arguments: bowtie -n 1 -m 1 -k 1 --trim5 5 -l 36 --best
ChIP-seq: Conversion to sorted BAM format using samtools
ChIP-seq: Peak calling using MACS2 version 2.0.10.20130306 with the following essential command line arguments: macs2 callpeak –bw 100 –m 5 50 –keep-dup 1 --qvalue=0.01 --bgd
Genome_build: rn4
Supplementary_files_format_and_content: gtf: standard gtf as produced by Cuffcompare
Supplementary_files_format_and_content: differential expression: standard gene_exp.diff files produced by Cuffdiff
Supplementary_files_format_and_content: endocePeak: MACS2-native format that is otherwise like the standard ENCODE-style narrowPeak format except that the 5th column contains transformed p-values (-log10pvalue*10)

Submission date

Jan 07, 2014

Last update date

May 15, 2019

Contact name

Sami Heikkinen

E-mail(s)

sami.heikkinen@uef.fi

Organization name

University of Eastern Finland

Department

Institute of Biomedicine