|
| Status |
Public on Jun 12, 2017 |
| Title |
48h_INTS12_D-siRNA_C_1 |
| Sample type |
SRA |
| |
|
| Source name |
48h_INTS12_D-siRNA_C
|
| Organism |
Homo sapiens |
| Characteristics |
donor: 195307
cell type: Human bronchial epithelial cells HBECs
passage: 3
source: Lonza
transfected with: INTS12_D-siRNA_C
time point: 48h since the initiation of knockdown
|
| Treatment protocol |
RNAseq: Effects of INTS12 depletion were assessed 120h and 48h after the initiation of interference.There were four experimental conditions: un-transfected cells, cells transfected with scrambled D-siRNA control, and cells transfected with D-siRNAs A and C. Each experimental condition was performed in three independent biological replicates.
ChIP-seq: Cells were formaldyhade fixed. Chromatin was isolated by the addition of lysis buffer. Genomic DNA for each replicate sample was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation.
|
| Growth protocol |
Human bronchial epithelial cells HBECs were purchased from Clonetics-Biowhittaker (MD, USA). Cells were cultured in HBEC basal medium (BEGM) from Lonza (Berkshire, UK) prepared by addition of all the recommended supplements per manufacturer specifications excluding gentamicin. All laboratory experiments were performed using passage 3 cells. Prior to experiments cells were grown at 37oC with 5% CO2 until ~95% confluent with BEGM media change every ~48h
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAseq: Total RNA was extracted using a mammalian total RNA prep kit with on-column DNaseI digestion
ChIP-seq: Unprecipitated genomic DNA (i.e. input control) was prepared from a pool of equal aliquots of the two donor samples. Genomic DNA regions of interest were isolated using 4 µg of antibody against INTS12 (Sigma cat. num. HPA03577) following manufacturer’s specifications (Active Motif).
RNA-seq: The sequencing libraries were prepared with Illumina TruSeq RNA Sample Prep Kit v2. mRNA was poly-A selected by capturing total RNA samples with oligo-dT coated magnetic beads. The mRNA was then fragmented and randomly primed. cDNA was synthesised using random primers. Finally a ready-for-sequencing library was prepared by end-repair, phosphorylation, A-tailing, adapter ligation and PCR amplification. Paired-end sequencing was performed on the HiSeq2000 platform (Illumina) using TruSeq v3 chemistry over 100 cycles yielding ~40 million reads per sample.
ChIP-seq: Libraries (Illumina) were prepared from the both ChIP and input DNAs and the resulting libraries were sequenced yielding ~40 million reads per two ChIP samples from each donor cells and one input control of both donors. DNA libraries obtained from single donor were sequenced on NextSeq 500 sequencing machine
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
processed data file: 48h_metaMatrix.txt
C1
|
| Data processing |
RNAseq alighnemnt performed using TopHat
RNAseq transcriptome assembly peroformed using Cufflinks
RNAseq differential gene expression performed using Cuffdiff (significant defined as q<0.05)
FPKM abundance per gene performed using Cuffnorm
ChIPseq alighnment performed with Bowtie2
ChIPseq peak calling perfmed with MACS2 (ChIP vs input control approach)
ChIPseq track signal performed by comparing ChIP vs input with fold enrichment option and total library size normalization. Generated bedGraphs were converted to wig files with 100bp resolution.
Genome_build: hg19
Supplementary_files_format_and_content: *metaMatrix.txt contains FPKM abundances per gene.
Supplementary_files_format_and_content: *.wig contains library size and background noise normalized signal tranck
Supplementary_files_format_and_content: *.narrowPeak contains inferred INTS12 binding sites at q<0.05
|
| |
|
| Submission date |
Apr 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander Kheirallah |
| E-mail(s) |
akk43@cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
Stem Cell Institute
|
| Street address |
Hills Road
|
| City |
Cambridge |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB2 0AH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE80386 |
Integrator complex subunit 12 is a key regulator of human protein synthesis pathways |
|
| Relations |
| BioSample |
SAMN04867485 |
| SRA |
SRX1711340 |
