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Sample GSM2247489

Query DataSets for GSM2247489

Status

Public on Mar 06, 2017

Title

Control replicate 3

Sample type

SRA

Source name

T-ALL cells

Organism

Homo sapiens

Characteristics

overexpressing or control: control
experiment number: 3
passage: 4

Treatment protocol

T-ALL Cells were plated in 24 well tissue culture treated plates (300,000 cell/ well) in lentiviral supernatant (MOI=1) mixed with 300 microliters of culture medium/ well. After 24 hours an additional 300 microliters of culture medium was added. After 48 hours of exposure to lentivirus, the cells were washed with DPBS and then replated in 500 microliters of culture medium. After a further 24 hours, transduced (GFP+) cells were sorted for RNA-Seq (n=3 experiments).

Growth protocol

Primagraft T-ALL cells (xenografted primary T-ALL cells) were generated by serial transplantations of deidentified bone marrow cells from a pediatric patient with relapsed T-ALL in NOD/SCID/IL2Rγ-/- mice. Primagraft T-ALL cells were cultured in alpha-MEM, 20% FBS, L-glutamine (2 mM), and Penicillin-streptomycin (0.5X). Primagraft cells at different passages of culture were transduced with a BCL11B expression or a control lentiviral vector (MOI=1), and 72 hours later, GFP+ cells were isolated by FACS for RNA-Seq.

Extracted molecule

total RNA

Extraction protocol

The Trizol method (Mirneasy RNA extraction mini kit, Qiagen, Valencia, CA) was used to extract total RNA from GFP+ T-ALL cells isolated by FACS at 72 hours post transduction.
The Ovation Human FFPE RNA‑Seq System (NuGen) was used to convert 25 ng of total RNA from GFP+ T-ALL cells into amplified cDNA libraries. The cDNA was sheared using a S220 focused ultrasonicator (Covaris, Woburn, MA) to generate an average fragment size of 300 base pairs. twenty cycles of PCR were used to ampify the libraries.
Libraries were sequenced on Illumina Nextseq500 (paired end 75 base pair sequencing)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Read alignment: Tophat v2.0.14 was used to align paired end reads to the human transcriptome and genome (hg 19)
Estimation of gene level counts: HTseq v0.6.1p1, whole genome annotation file of protein coding and long non-coding RNA genes published in Casero et al, Nature Immunology 2015 (PMID: 26502406) was used for the Htseq analysis.
Differential expression analysis: DESeq2, experiment identifier (experiment number 1, 2 or 3) was used as a covariate
Genome_build: hg19
Supplementary_files_format_and_content: counts

Submission date

Jul 21, 2016

Last update date

May 15, 2019

Contact name

chintan parekh

E-mail(s)

cparekh@chla.usc.edu

Organization name

Children's Hospital Los Angeles

Department

Pediatrics