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Sample GSM4717168

Query DataSets for GSM4717168

Status

Public on Mar 31, 2021

Title

HEK293T-25ng-NAIN3-rep1

Sample type

SRA

Source name

HEK293T cell line

Organism

Homo sapiens

Characteristics

cell line: HEK293T

Treatment protocol

For structure probing, cells were treated with NAI-N3 or DMSO at 37°C for 5min and quenched by adding 500 µl Trizol. For RNA seq, cells were treated with 100 ng/ml LPS for 0 h or 3 h before RNA isolation. For uvCLAP, cells were maintained in media supplied with 50 µM biotin overnight and treated with 100 ng/mL LPS for 3 h.

Growth protocol

HEK293T cells, iBMDM and Raw264.7 cells were cultured were maintained in DMEM (Gibco) medium with high glucose, supplemented with 10% FBS and 1% Penicillin-Streptomycin at 37°C with 5% CO2.

Extracted molecule

polyA RNA

Extraction protocol

For smartSHAPE and RNA-Seq, RNAs were isolated with Trizol reagents.
For smartSHAPE, cells were treated with NAI-N3 or DMSO and RNAs were isolated with Trizol followed by rRNA removal. RNAs were reverse transcribed with random primers, and RNase I digestion was performed at 37 °C for 30 min. Enrichment was performed using MyOne C1 beads. After elution, cDNAs were ligated with biotinylated 3’ adaptor using CircLigase and immobilized with MyOne C1 beads. Second strand synthesis and 5’ligation was performed on the beads. smartSHAPE libraries were generated by amplification of ligation products. RNA seq libraries of Reg1 KO and WT iBMDMs were generated with KAPA RNA Hyper Prep kit. uvCLAP libraries were generated as published protocol (PMID: 29559621).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Description

HEK293_25ng.shape.txt

Data processing

Library strategy: smartSHAPE
Calculate smartSHAPE score from smartSHAPE sequencing data:
1) The 3’ adaptor is removed with Cutadapt
2) Duplicated reads were collapsed with custom script;
3) The first 10nt were removed using trimmomatic
4) Clean reads were mapped to human rRNA with Bowtie2
5) Un-mapped reads were mapped to the human (hg38) or mouse (mm10) genome using STAR
6) Sam files were convert into .tab files using icSHAPE-pipe sam2tab;
7) The smartSHAPE score was calculated using icSHAPE-pipe calcSHAPENoCont
For uvCLAP data:
Raw reads were trimmed by Cutadapt and collapsed to remove duplicated reads. The clean reads were mapped to mouse rRNA by Bowtie2, and the remaining un-mapped reads were re-mapped to mouse genome (mm10) by STAR. Peaks were called out by Piranha with Reg1 group as response sample and GFP group as covariate sample. The bin size was set to 50 with -z 50.
For RNA-Seq data:
Raw reads were trimmed by Cutadapt. The trimmed reads were mapped to mouse rRNA by bowtie2, and the remaining un-mapped reads were re-mapped to mouse genome (mm10) by STAR. We set parameter --quantMode TranscriptomeSAM GeneCounts to generate the read count file. The RNA-Seq differentail expression analysis is carried by DESeq2.
Genome_build: hg38 and mm10
Supplementary_files_format_and_content: .shape format: tab-seperated file, each row represent a transcript. The meaning of each column: transcript_id, length, a star symbol, reactivity score for each base

Submission date

Aug 10, 2020

Last update date

Mar 31, 2021

Contact name

Meiling Piao

E-mail(s)

pml16@mails.tsinghua.edu.cn

Organization name

Tsinghua University

Department

Life Science