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Sample GSM2464652

Query DataSets for GSM2464652

Status

Public on Mar 06, 2017

Title

Knockdown CD1a- replicate 2

Sample type

SRA

Source name

Knockdown CD1a-

Organism

Homo sapiens

Characteristics

cell type: Cord blood CD34+ cells
transduced with: BCL11B knockdown shRNA lentivral vector containing a GFP reporter
cell populatione: CD45+GFP+CD7+CD1a- T-cell precursors
experiment number: 2

Treatment protocol

CD34+ enriched cord blood cells (enriched by magnetic activated cell sorting) were cultured for 16 hours in 100 microliters of transduction medium (X-Vivo 15 [Lonza, Walkersville, MD], thrombopoietin [50ng/ml], FLT3 ligand (50 ng/ ml], Stem cell factor [50 ng/ ml], and l-glutamine [2 mM, Cellgro, Manassas, VA]) on retronectin (50 ng/ ml, Clontech) coated non tissue culture treated 48 well plates (100,000 cells/ well). ShRNA Lentiviral supernatant (multiplicity of infection=10) along with additional transduction medium (to achieve a 1:1 volume ratio for supernatant and transduction medium) was then added. 125 microliters of transduction medium was added 24 hours after the dose of lentivirus. After 48 hours of exposure to lentivirus, CD34+GFP+lin(CD3, CD4, CD8,CD19)- cells were sorted for co-culture with OP9DLL1 stroma.

Growth protocol

CD34+ cord blood (CB) cells were transduced with knockdown or scrambled control shRNA (multiplicity of Infection [MOI]=10) lentivirus. CD34+GFP+lin- CB cells were sorted (fluorescence activation cell sorting, FACS) and then co-cultured with OP9DLL1. The medium for OP9DLL1 co-cultures medium consisted of alpha-MEM (ThermoFisher), 20% fetal bovine serum (FBS, Hyclone, lot no. AXF42576), L-glutamine (2 mM, Cellgro, Manassas, VA), Pencillin-streptomycin (0.5X, CellGro), IL-7 (5 ng/ ml), and Flt3 ligand (5 ng/ml). On day 14 of co-culture, CD45+GFP+CD7+CD1a- and CD45+GFP+CD7+CD1a+ T-cell precursors were isolated from control and knockdown co-cultures by FACS and analyzed by RNA-Seq to determine the effect of BCL11B knockdown on the transcriptome of T-cell precursors.

Extracted molecule

total RNA

Extraction protocol

The Trizol method (Mirneasy RNA extraction mini kit, Qiagen, Valencia, CA) was used to extract total RNA from CD45+GFP+CD7+CD1a- and CD45+GFP+CD7+CD1a+ T-cell precursors isolated by FACS on day 14 of cord blood cells-OP9DLL1 co-cultures .
The Ovation Human FFPE RNA‑Seq System (NuGen) was used to convert 25 ng of total RNA from T-cell precursors 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

Description

Sample 7

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: Raw counts for genes (HTseq output)

Submission date

Jan 21, 2017

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