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Sample GSM7757702

Query DataSets for GSM7757702

Status

Public on Mar 20, 2024

Title

KYSE-150_NSUN6_KD_input

Sample type

SRA

Source name

esophagus

Organism

Homo sapiens

Characteristics

tissue: esophagus
cell line: KYSE-150
cell type: esophageal epithelial cells
genotype: NSUN6-knockdown

Treatment protocol

KYSE-150 cells were transfected with shRNA for stable knockdown of NSUN6. KYSE-150 cells with normal expression of NSUN6 were used as control.

Growth protocol

KYSE-150 cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 1% streptomycin (Gibco, USA) and 1% Penicillin (Gibco, USA). Cells were cultured in a water-saturated atmosphere under 5% CO2 at 37 °C in an incubator (Thermo Scientific, USA).

Extracted molecule

polyA RNA

Extraction protocol

Cells were treated with 100μg/ml cycloheximide and then incubated with 1ml cell lysis buffer [1% Triton X-100 in ribosome buffer (RB buffer) [20mM HEPES-KOH (pH 7.4), 15mM MgCl2, 200mM KCl, 100μg/ml cycloheximide and 2mM dithiothreitol] on ice. 30 minutes later, cell lysates were collected and centrifuged at 16,200g for 10 minutes at 4℃. 10% of the supernatants was used as input control.The remaining supernatants was layered onto 35ml sucrose buffer (30% sucrose in RB buffer) and then subjected to after ultra-centrifugation at 174,900g for 5 hours at 4℃ in a SW32 rotor (Beckman Coulter, USA) to collect the mRNAs. Purified RNA samples were isolated from the input and polyribosome-bound mRNAs using TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions for subsequent library construction and RNA-seq.
Libraries were prepared according to Illumina's instructions accompanying the TrueSeq Stranded mRNA library preparation kit.
Polyribosome-bound mRNAs sequencing (polyribosome-seq)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

sh-input_FPKM

Data processing

Reads were aligned to hg19 using tophat
High quality reads were mapped to UCSC human reference genome(GRCh37/hg19). The gene expression level (Columns 2 to 4 in the txt file) was normalized by using FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method. Translational efficiency (TE Column 5 in the txt file) were calculated using the following formula: TE = (FPKM in RNC)/(FPKM in input).
Assembly: hg19
Supplementary files format and content: txt

Submission date

Sep 05, 2023

Last update date

Mar 20, 2024

Contact name

hui han

E-mail(s)

hanh23@mail2.sysu.edu.cn

Organization name

the first affiliated hospital，sun yat-sen university

Street address

yuexiu