|
| Status |
Public on Oct 01, 2021 |
| Title |
scrambled_donor3 |
| Sample type |
SRA |
| |
|
| Source name |
human naïve CD4 T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: human naive CD4 T cells
genotype/variation: control siRNA (scrambled)
|
| Treatment protocol |
Human naïve CD4 T cells were transfected by electroporation with 2 uM JMJD3-specific Cy3-labeled siRNA (ON-TARGETplus siRNA smartpool, GE Dharmacon) or Cy3-labeled scramble siRNA controls after isolation and incubated in culture for 16 hrs. To isolate the cells being efficiently transfected, Cy3-positive populations were sorted by flow cytometry in both siJMDJ3 and scramble transfected cells. These populations were then activated with CD3/CD28 beads and collected at certain time points after activation for downstream experiments.
|
| Growth protocol |
Naïve CD4 T cells were cultured in RPMI 1640 (Gibico) supplemented with 100 U/ml Penicillin, 100 ug/ml Streptomycin and 10% FBS at 37° C with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA for RNA-seq was isolated from purified cells using an RNeasy plus mini kit (QIAGEN) following the manufacturer’s instructions.
200 ng of purified total RNA was put into sequencing library prep, using NEBNext® Ultra™ RNA Library Prep Kit and for Illumina®NEBNext rRNA Depletion Kit (NEB). Libraries were assessed on an Agilent Bioanalyzer using a DNA chip and quantitated using the Quant-iT ds DNA BR Assay kit (Invitrogen) and a Qubit Fluorimeter (Invitrogen).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
RNA-seq reads were aligned to the UCSC hg38 genome using Tophat 2
Reads assigned to each gene were counted by subread with ensembl gene sets as reference.
Genes without at least 5 reads mapped in at least one sample were considered undetectable and were filtered out.
Read counts were normalized by TMM method and differential expression was calculated using R package edgeR Cut-offs imposed for differential expression analysis was set as FDR of < 0.1.
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited files. Output of differential gene expression analysis from EdgeR.
|
| |
|
| Submission date |
Oct 11, 2018 |
| Last update date |
Oct 01, 2021 |
| Contact name |
Xiangzhi Meng |
| E-mail(s) |
mengxiangzhi919@gmail.com
|
| Organization name |
The University of Hong Kong
|
| Street address |
21 Sassoon Road
|
| City |
Hong Kong SAR |
| ZIP/Postal code |
000000 |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE121108 |
Gene expression analysis on Naïve CD4 T cell with JMJD3 knockdown |
|
| Relations |
| BioSample |
SAMN10230579 |
| SRA |
SRX4834246 |
