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Sample GSM2297023

Query DataSets for GSM2297023

Status

Public on Dec 28, 2022

Title

siTotal1_1

Sample type

SRA

Source name

HCT116 colon cancer cell line

Organism

Homo sapiens

Characteristics

knockdown: siRNA #1 to total NFYA

Treatment protocol

Custom siRNAs targeting all NFYA transcripts (siTotal; ACUAGAGGCAGAAGGGAAA; UGGUGAAGGUGGACGAUUU), NFYA transcripts with the longest 3’ UTR in RefSeq (siLong; AGUGAUCAUUGUAGGGUAA; GGUCAUUCCUUUAGCAUCA), or no known mammalian transcripts (siGENOME non-targeting siRNA #3; D-001210-03-05) were purchased from Dharmacon. For NFYA siRNAs, sequences were input into Dharmacon’s custom siRNA website as the sense strand, and then the website populated the anti-sense strand and added UU overhangs to the 3’ end of each strand. HCT116 cells were seeded at 25,000 cells per well of a 6-well plate, then transfected after 24 hours with siRNAs. Transfection for a single well of a 6-well plate was as follows: 250 µL of OptiMEM (Thermo Fisher Scientific) was mixed with 1.25 µL of 100 µM siRNA, and then mixed with 250 µL OptiMEM containing 5 µL Lipofectamine 2000 (Thermo Fisher Scientific). siRNA:Lipofectamine complexes were formed by incubation at room temperature for 20 minutes, and then all 500 µL of the siRNA:Lipofectamine mixture added to a well containing 1.5 mL of media, for a final siRNA concentration of 62.5 nM. 72 hours after siRNA transfection, cells were harvested (approximately 75% confluence) by PBS wash, scraping into PBS, pelleting at 500g for 2 minutes, and storage of cell pellet at -80°C.

Growth protocol

HCT116 cells were grown in McCoy's 5A (Modified) Medium (Thermo Fisher Scientific) with 10% FBS (Atlanta Biologicals) at 37°C and 5% CO2.

Extracted molecule

polyA RNA

Extraction protocol

Total cellular RNA was prepared from each cell pellet using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer’s instructions.
RNA-seq libraries were prepared by Genome Quebec using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Read alignments were performed using STAR version 2.4.0f1. Read counts were tabulated for ENSEMBL genes build 75 using the featureCounts() function from the R package Rsubread (version 1.20.6) with options useMetaFeatures=TRUE, isPairedEnd=TRUE, nthreads=18, minMQS=30, requireBothEndsMapped=TRUE, checkFragLength=FALSE, countChimericFragments=FALSE, strandSpecific=2.
Genome_build: hg19
Supplementary_files_format_and_content: A single processed file contains one row for each gene in ENSEMBL genes build 75. There is one column for each sample in this record. Each data value is the raw, un-normalized counts value reported by featureCounts(). Each gene is denoted by the ENSEMBL gene ID in the first column.

Submission date

Aug 29, 2016

Last update date

Dec 28, 2022

Contact name

Angela H Ting

E-mail(s)

ahting@mdanderson.org

Organization name

The University of Texas MD Anderson Cancer Center

Department

Epigenetics and Molecular Carcinogenesis