|Public on Nov 22, 2019
age: 62 years
|For fetal iTreg cells,CRISPR-Cas9 editing via nucleofection of Cas9 complex with the Helios gRNA1 (HeliosKO) or non-targeting guide (HeliosWT) was carried out prior to Treg differentiation
|Treg differentiation - Cells were stimulated with anti-CD3/CD28/CD2 tetratmers (Stemcell) in culture media supplemented with with 10ng/mL IL-2 (Peprotech) in the presence of absence of 50ng/mL TGFb (Peprotech)
|Cells were harvested on Day 6 of culture, and total RNA was extracted from 25,000 cells using the Nucleospin XS RNA kit (Machery Nagel)
Libraries were constructed using a standard Illumina protocol following manufactorer instructions using the NEBNext® Ultra™ II Directional RNA Library Prep Kit
|Illumina HiSeq 4000
|Sequencing batch 1
|Fastq files were mapped to hg19 with Bowtie 2
Read counts per gene was obtained using RSEM
TMM normalization and differential expression analysis performed using EdgeR package for Bioconductor
Separate comparisons were performed for adult and fetal iTreg cells, and fetal HeliosKO and HeliosWT iTreg cells using generalized linear models
TMM read counts for HeliosKO versus HeliosWT comparisons are normalized for paired samples and batch effects due to two sequencing runs carried out, and are supplied as log2 read counts.
Supplementary_files_format_and_content: Generalized linear model output with fold change and FDR in text files for both comparisons. TMM normalized read counts for both comparisons in txt files.
|May 21, 2019
|Last update date
|Nov 24, 2019
|University of California, San Francisco
|35 Medical Centre Way, Regeneration Medicine Building 903B
|Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells.
|Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells [RNA-Seq - induced Treg cells].