|
| Status |
Public on Jul 23, 2022 |
| Title |
RNAseq-KO2 CD4 T cell-0h |
| Sample type |
SRA |
| |
|
| Source name |
Primary CD4 T cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: spleen
age: 8 weeks
genotype: Trmt61a cKO
cell line: primary CD4 T cells
treatment: Anti-CD3(5ug/ml)/CD28(2ug/ml)
|
| Treatment protocol |
The purified T cells were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) in replicate wells of 96-well plates.
|
| Growth protocol |
Primary T cells were isolated from the spleens of age-matched WT and conditional knockout mice (8 weeks old). Naive CD4 T cells were purified by using EasySep Mouse Naïve CD4+ T Cell Isolation Kit (STEMCELL Technologies) according to the instructions, respectively. The purified T cells were cultured with X-VIVO mediums.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAseq: total RNA was extracted based on the manufacturer’s protocol by using TRIzol reagent (Invitrogen). Riboseq: we performed the ribosome profiling by strictly following the manual for the Illumina TruSeq Ribo Profile (Mammalian) Kit. m1Aseq: 200 ng small RNA fraction (<200 nt; mainly composed of mature tRNA) was purified from total RNA using MEGAclear Kit (Thermo Fisher Scientific).
RNAseq:RNA libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB) based on the manufacturer’s instructions. RiboTagseq: libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (no. E7300S, E7300L). m1Aseq: Purified small RNA was deacylated by incubating in 0.1 M Tris-HCl, pH 9 at 37°C for 45 min. Half of the deacylated small RNA fraction was subjected to demethylation treatment by using AlkB in vivo reaction. RNA samples were dephosphorylated with PNK (NEB) and ligated to 3’ RNA linker using T4 RNA ligase2, truncated KQ (NEB). Reverse transcription was carried using different reverse transcriptase for different samples. 5’ adaptor was ligated to cDNA and then amplified by PCR. PCR products were purified by 8% TBE gel.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
total RNA
|
| Data processing |
Reads adapters were removed by Trim_galore ( v0.6.6)
For RNA-seq, Index of the reference genome was built using Hisat2 (v2.0.5) and paired-end clean reads were aligned to the mm10 genome using Hisat2 (v2.0.5) ,and then featureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. For Ribo-seq, the rRNA removed reads of each sample were mapped to the reference genome by Bowtie2(V 2.4.1) allowing no mismatches. For m1A-seq, clean reads were mapped to mm10 tRNA genome by bowtie2.
Genome_build: mm10 genome;mm10 tRNA reference
|
| |
|
| Submission date |
Sep 28, 2021 |
| Last update date |
Jul 23, 2022 |
| Contact name |
Xiaoyu Li |
| E-mail(s) |
xiaoyu_li@zju.edu.cn
|
| Organization name |
Zhejiang University
|
| Department |
SCHOOL OF BASIC MEDICAL SCIENCES
|
| Lab |
Li Lab
|
| Street address |
866 Yuhangtang Road
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE184909 |
tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis |
|
| Relations |
| BioSample |
SAMN21880101 |
| SRA |
SRX12384007 |
