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Sample GSM6928211

Query DataSets for GSM6928211

Status

Public on Sep 07, 2023

Title

L31423_K562_endogenous_RNA_IFNA_replicate3

Sample type

SRA

Source name

K562

Organism

Homo sapiens

Characteristics

cell line: K562
cell type: Lymphoblast cells
genotype: wildtype
treatment: IFNA

Treatment protocol

In this data set, 6 replicate flasks of K562 cells were treated for 6 hours with each of the following treatments: dexamethasone (1 uM), IFNA2b (2,000U/ml), or vehicle control (media). Cells were then collected for mRNA-Seq to assess the endogenous gene expression response of K562 cells to treatment.

Growth protocol

We made 18 replicate T75 flasks, each containing 12 million K562s. 42 hours later, K562s were stimulated with treatment, and cells were collected 6 hours post-treatment.

Extracted molecule

polyA RNA

Extraction protocol

We extracted RNA from the separately aliquoted cells using the Qiagen RNEasy kit.
We prepared RNA-Seq libraries using the NEBNext Ultra II RNA Library Prep Kit for Illumina.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

L31423

Data processing

Reads were trimmed with Trim Galore (Trim Galore version 0.6.4_dev; Cutadapt version 2.3) to remove basepairs with a Phred score less than 20, and end sequences that matched at least two basepairs of the adapter sequence. Only trimmed reads longer than 25 basepairs were retained.
We used the STAR package (version 2.5.0) (Dobin et al., 2013) two-pass mapping to map the filtered reads to the hg38 genome. We retained uniquely mapped reads by filtering the output SAM file to keep reads with MAPQ = 255.
We then used htseq (version 0.6.0) (Anders et al., 2014) to quantify read counts per gene.
We only retained genes that had TPM > 3 in at least 3 of 6 samples in at least one of the three conditions (baseline, IFNA, or dex).
Only protein-coding genes were retained for final analysis, resulting in a total of 10,676 testable genes.
Assembly: hg38
Supplementary files format and content: Tab delimited text file with data matrix of raw read counts mapping to a gene, where each row represents a gene, and each column represents a biological replicate (i.e., library). Only protein-coding genes that had transcripts per million > 3 in at least 3 of 6 samples in at least 1 of the 3 conditions (baseline, IFNA, or dex) are included.

Submission date

Jan 11, 2023

Last update date

Sep 07, 2023

Contact name

Rachel A Johnston

E-mail(s)

racheljohnston7@gmail.com

Organization name

Duke University

Department

Evolutionary Anthropology