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Sample GSM3984255

Query DataSets for GSM3984255

Status

Public on Nov 21, 2019

Title

KO_RNase_Riboseq_rep1

Sample type

SRA

Source name

HEK293T

Organism

Homo sapiens

Characteristics

genotype: PUS10 -/-
cell line: HEK293T
molecule subtype: Ribosome protected RNA

Treatment protocol

For cell passage, cells were first washed with PBS. Trypsin was added to the dishes, which were then incubate at 37 °C for 2-5 min, and FBS containing medium was used to inactive trypsin. After collected by centrifuge at 1000 g for 3 min, cells were counted and split for different purposes. Generation of PUS10 knockout HEK293T cells was achieved by using CRISPR/Cas9 technology.

Growth protocol

HEK293T cells were maintained at 37 °C in DMEM medium (Corning) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco).

Extracted molecule

total RNA

Extraction protocol

For rRNA(-) nuclear RNA-seq, cell fractionation was performed, and the resulting RNA was subjected to two rounds of rRNA removal using RiboMinus™ Eukaryote Kit for RNA-Seq .For polyA RNA-seq, total RNA was isolated from cell lines with Trizol (invitrogen) according to the manufacturer’s instructions. Two rounds of poly-A selection were performed using Dynabeads™ Oligo(dT)25 (Invitrogen) to obtain mRNA. For PAR-CLIP-seq, five 15 cm dishes of HEK293T cells were transfected with flag-tagged PUS10 plasmid at 80% confluency. After 6 h, the media was changed and 200 μM 4SU was added. After additional 18 h, the cells were washed once with 10 ml ice-cold PBS for each plate. Then, the plates were kept on ice, and the UV crosslink was carried out twice by 150 mJ/cm2 at 365 nm. After immunoprecipitation, the final recovered RNA sample was further cleaned by RNA Clean & Concentrator (Zymo Research). For DM-Ψ-seq, small RNA (<200 nt) was isolated and purified from cell culture using miRNeasy Mini Kit, RNeasy MinElute Cleanup Kit and RNase-free DNase Set (Qiagen) according to the manufacturer’s instructions.
The rRNA(-) nuclear RNA and poly-A RNA were used to construct RNA-seq library under the instruction of NEBNext® Ultra™ RNA Library Prep Kit for Illumina. For PAR-CLIP-seq, the RNA samples were used to construct library under the instruction of NEBNext® Multiplex Small RNA Library Prep Set for Illumina. For DM-Ψ-seq, the library construction was performed according to the eCLIP library construction protocol with several modifications.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Data processing

Library strategy: Ribo-seq
Basecalls performed using Illumina CASAVA.
For reads cleanning, adaptor were trimmed and low quality reads were filtered with Trim_galore software (version: 0.3.3).
Cleaned reads were mapped to reference genome (hg19 and genomic tRNA database [GtRNAdb (http://gtrnadb.ucsc.edu/genomes/eukaryota/Hsapi19/)]) using TopHat version 2.1.0 (rRNA(-) nuclear RNA-seq and polyA RNA-seq) and Bowtie version 1.2.2 (PAR-CLIP-seq and DM-Ψ-seq).
Abundance measurements were performed by Cufflinkes version 2.2.1 (polyA RNA-seq) and Htseq-count version 0.9.1 (rRNA(-) nuclear RNA-seq). Peak-calling of PAR-CLIP were performed by PARalyzer version 1.5. Site-calling of DM-Ψ-seq were performed by custom scripts.
Genome_build: hg19
Supplementary_files_format_and_content: Codon_Occupancy.tar
Supplementary_files_format_and_content: MNase_TE.txt

Submission date

Jul 29, 2019

Last update date

Nov 21, 2019

Contact name

Jinghui Song

E-mail(s)

[email protected]

Organization name

Peking university

Street address

5 Yiheyuan Road, Haidian District