|
| Status |
Public on Dec 20, 2016 |
| Title |
HCT116_NS_shRNA_1 - RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
Colorectal cancer cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
genotype/variation: Control
|
| Growth protocol |
Sections of snap-frozen tissues were either micrococcal nuclease treated or cross-linked with 1% formaldedyde for 20 minutes and sonicated for ChIPseq experiments followed b ChIP with 10ug of antibody. HCT116 cells were cross-linked with 1% formaldehyde for 20 min at 37oC, followed by ChIP with 10 µg CNOT3 for ChIPseq. For RNAseq experiments cells were washed with PBS and total RNA was extracted with TRIzol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA was treated with the Turbo DNA-free kit (Ambion) to remove genomic.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
ChIPseq: Lysates were clarified from micrococcal nuclease digested or sonicated or and histone-DNA or CNOT3-DNA complexed were isolated with antibody.
RNAseq: Total RNA was extracted with TRIzol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA was treated with the Turbo DNA-free kit (Ambion) to remove genomic DNA.
For ChIPseq:ThruPLEX-FD kits (Rubicon Genomics) was used and 50 bp single-end reads were sequenced on a HiSeq2000 instrument (Illumina).
For RNAseq: TruSeq RNA Sample Preparation Kit (Illumina) was used according to the manufacturer's instructions and 75 bp single-end reads were sequenced on an Illumina NextSeq 500 instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Control_1
RNAseq
Total RNA was extracted with TRIzol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA was treated with the Turbo DNA-free kit (Ambion) to remove genomic DNA. RNA-seq libraries were prepared from 300 ng RNA using the TruSeq RNA Sample Preparation kit (Illumina) and 75-bp single-end sequences were obtained on a NextSeq 500 Instrument (Illumina).
|
| Data processing |
Alignment: For ChIPseq experiments we used Bowtie2 (2.1.0) with settings (--no-unal). For RNAseq we used Tophat, version 2.0.9, default options.
ChIPseq analyses: For peak calling we used MACS2 using Q:0.01.
RNAseq analyses: normalized and differential expression: Cufflinks, version 2.2.1; settings: -b, -u, and default; Cuffmerge was used with reference gtf and default settings; Cuffquant was used with default settings; Cuffnorm was used with default settings; Cuffdiff was used with -b, -u and default settings.
Genome_build: Hg19
Supplementary_files_format_and_content: ChIPseq: Wiggle files were generated by NPS software using parameters by default. Bed files (the 5 columns are 1: chromosome, 2: start location, 3: end location, 4: peak number, 5: score).
Supplementary_files_format_and_content: RNAseq: Individual samples have an FPKM gene tracking file from Cufflinks, estimating transcript abundance for that sample, along with confidence intervals. There are two processed data matrices that apply to all the samples. There exists a differential expression table, prepared using Cuffdiff with an FDR of 5%, which contains average normalized FPKM values for Controls infected with a non specific NS_shRNA construct (Scr) and CNOT3-depleted by infection with shRNA_#3 construct (silenced), as well as fold change in expression, P-values, and Q-values corrected for multiple hypothesis testing.
|
| |
|
| Submission date |
Feb 23, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Paloma Cejas |
| E-mail(s) |
paloma_cejas@dfci.harvard.edu
|
| Phone |
6178406690
|
| Organization name |
Dana Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Street address |
450 Brookline Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE66216 |
Nucleosome dynamics in human colorectal cancer specimens reveal activation of a CNOT3-regulated pathway of embryonic stem cell self-renewal |
|
| Relations |
| BioSample |
SAMN03366675 |
| SRA |
SRX886406 |
