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Sample GSM2871676

Query DataSets for GSM2871676

Status

Public on Dec 02, 2017

Title

CD274_1

Sample type

SRA

Source name

glioblastoma cell line U251

Organism

Homo sapiens

Characteristics

cell line: U251
treatment: stable transfected cell line with PD-L1 overexpression
vector: Lentivirus
passage: 5-10

Growth protocol

U251/PD-L1 or U251/Vec were normally cultured in DMEM supplemented with 10% FBS. And 12 h after the passage, 6 culture dished were subject to total RNA isolation.

Extracted molecule

total RNA

Extraction protocol

Total RNA were extracted. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Basecalls performed using CASAVA version 1.8
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using STAR and paired-end clean reads were aligned to the reference genome using STAR (v2.5.1b). STAR used the method of Maximal Mappable Prefix(MMP) which can generate a precise mapping result for junction reads
HTSeq v0.6.0 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Genome_build: hg19
Supplementary_files_format_and_content: Text files includes raw readcounts for all samples or normalized readcounts for all samples.

Submission date

Dec 01, 2017

Last update date

May 15, 2019

Contact name

Xin Yao Qiu

E-mail(s)

[email protected]

Phone

+86-18040544911

Organization name

Huazhong University of Science and Technology