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Status |
Public on Jul 23, 2022 |
Title |
RiboTag-WT_IP_Rep2 |
Sample type |
SRA |
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Source name |
spleen
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Organism |
Mus musculus |
Characteristics |
tissue: spleen cell type: primary CD4 T cells genotype: Trmt6-WT (RiboTagflox/floxCd4Cre) treatment: Anti-CD3(5ug/ml)/CD28(2ug/ml)
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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.
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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.
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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.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
BGISEQ-500 |
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Description |
For tRNA-seq, naïve CD4+ T cells were isolated from WT mice and activated with plate-bound anti-CD3 plus anti-CD28 in a 96-well plate for 0,6,18,48 hours. 200 ng small RNA fraction (<200 nt; mainly composed of mature tRNA) was purified from total RNA using MEGA clear Kit (Thermo Fisher Scientific). Purified small RNA was deacylated by incubating in 0.1 M Tris-HCl, pH 9 at 37°C for 45 minutes. For RiboTag-seq, 500 million naïve CD4+ T cells were isolated from WT (RiboTagflox/floxCd4Cre) and Trmt61a-KO (Trmt61aflox/floxRiboTagflox/floxCd4Cre) mice (activated with plate-bound anti-CD3 plus anti-CD28 in 96-well plate for 6 hours), and washed and treated with 100 μg/mL cycloheximide for 1 minute. Cells were washed in ice-cold PBS twice, and then RiboTag lysis buffer was added directly to the cells on ice as previously described. Cell lysates were passed through a 26-gauge needle 10 times and incubated for 30 min on ice to ensure complete lysis. Ribosome-RNA-containing supernatants were clarified by centrifugation at 12,000g for 10 min at 4 °C. Haemmaglutinin-conjugated magnetic beads (Pierce) were added to samples and incubated overnight under gentle inversion at 4 °C. Beads were washed three times for 10 min with gentle rotation in a high-salt buffer containing cycloheximide. RNA was eluted from HA-beads using Qiagen RLT buffer containing 2-Mercaptoethanol and anti-foaming DX reagent (Qiagen) by 30 s vortex pulsing. RNA was isolated using an RNAeasy micro kit. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples were transferred to the OE Biotech Co., Ltd. (Shanghai, China) for cDNA library preparation using NEBNext® Ultra II Directional RNA Library Prep Kit according to the manufacturer’s instructions.
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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. Assembly: mm10,mm10-tRNA Supplementary files format and content: tRNA expression in txt file format Supplementary files format and content: RiboTag-seq in bigwig file format Library strategy: RiboTag-Seq
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Submission date |
Jul 23, 2022 |
Last update date |
Nov 29, 2022 |
Contact name |
Xiaoyu Li |
E-mail(s) |
xiaoyu_li@zju.edu.cn
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Organization name |
Zhejiang University
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Department |
SCHOOL OF BASIC MEDICAL SCIENCES
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Lab |
Li Lab
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Street address |
866 Yuhangtang Road
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City |
Hangzhou |
State/province |
Zhejiang |
ZIP/Postal code |
310058 |
Country |
China |
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Platform ID |
GPL23479 |
Series (1) |
GSE184909 |
tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis |
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Relations |
BioSample |
SAMN29921454 |
SRA |
SRX16528666 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6380474_WT2_IP.sort.bw |
7.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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