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Sample GSM5202129

Query DataSets for GSM5202129

Status

Public on Mar 24, 2021

Title

Patient-derived GSC -BT12 shCTRL D6-

Sample type

SRA

Source name

Patient-derived glioblastoma stem cell

Organism

Homo sapiens

Characteristics

molecular subtype/phenotypic state: Mesenchymal/Mesenchymal
cell culture condition: serum-free DMEM/F-12 medium supplemented with 1 x B27 (both from Gibco), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 15 mM HEPES (all from Lonza), 0.02 μg/ml human EGF, and 0.01 μg/ml human FGF-basic.
cell transduction: scrambled short hairpin RNA, day 5 post silencing

Treatment protocol

Following shRNA constructs from the RNAi Consortium (TRC) library (Broad Institute of MIT and Harvard) in pLKO.1 lentiviral vector were used to silence CD109 expression: human shCD109#1 (TRCN0000073649). Complete targeting sequences were as follows (5’- 3’): shCD109; GCCGATCCTTACATAGATATT. Lentivirus was produced by transfecting shRNA constructs or non-targeting control vector (shControl) together with CMVg and CMV delta8.9 packaging plasmids (both from Addgene) and Fugene 6 transfection reagent (Promega) into 293FT cells. Silencing efficiencies of different shRNA constructs were confirmed using western blotting and/or qRT-PCR.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from lysed cells or gliospheres using the RNeasy Mini kit (#74104) (Qiagen) according to the manufacturer’s instructions. After DNase treatment, the quality of the RNA preparations (1 µg) was controlled using a Tapestation (Agilent). Total RNA 1 ug is treated with Illumina's Ribo-Zero Complete Gold Kit to remove the ribosomal RNA. First the samples are treated with rRNA removal solution after which the rRNA is removed combining probe-hybridized samples and magnetic beads. The Ribo-Zero treated RNA is purified with Qiagen RNeasy MinElute Cleanup Kit. The absence of rRNA is assessed by Bioanalyzer and the quantity of RNA by Bioanalyzer.
NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420S/L Version 8.0 3/16) is used to generate cDNA libraries for next generation sequencing. First, the ribosomal RNA depleted sample (10 ng) is fragmented to generate inserts around 200 bp, and then primed with random primers. The first strand cDNA synthesis utilizes Actinomycin D, which inhibits the DNA polymerase activity of the reverse transcriptase increasing strand specificity. In the second strand cDNA synthesis dUTP labelled oligo nucleotides are incorporated to mark the second strand with uracils (U). The cDNA synthesis product is purified with Agencourt AMPure XP beads. Next, the cDNA is end-repaired, and adapter ligated utilizing dA-tailing. The adaptor ligated DNA goes through a bead-based size selection after which the final PCR enrichment takes place. At this point, each sample is given a unique index to enable pooling of multiple samples (multiplexing) for sequencing. During the high-fidelity PCR, USER (Uracil-specific Excision Reagent) enzyme cuts away the uracil strand preserving only the first strand. In addition, the loop adaptor is cut open to enable the PCR. The amplified library is then purified using AMPure XP Beads. Library quality is assessed by Bioanalyzer (Agilent DNA High Sensitivity chip) and library quantity by Qubit (Invitrogen).
Illumina NextSeq 500 High Output 75 cycle kit for single-end 1x75bp reads (2 flow cells). Loading concentration 1,5 pM + 1% PhiX control. Starting amount of Total RNA: 1000 ng, PCR cycles used in method: 15.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

GSCs_RNAseq_1.xlsx
BT_12_SHCTRL_D5_S12

Data processing

bcl2fastq2 Conversion Software was used to convert BCL files to FASTQ file format and demultiplex samples.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using Trimmomatic. Parameters used: :2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Trimmed reads were mapped to GRCh38.p12 reference genome (for processed data file included in the GSCs_RNAseq_1.xlsx) and GRCh38.p13 from GENCODE human release v.33 (for processed data file included in the GSCs_RNAseq_2.xlsx) using STAR aligner (2.6.0c) with parameters --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimSegmentReadGapMax 3 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJstitchMismatchNmax 5 -1 5 5 --outFilterType BySJout --outFilterMismatchNmax 999 --outSAMattributes NH HI NM MD AS nM jM jI XS --outSAMtype BAM SortedByCoordinate --outSAMmapqUnique 60 --quantMode GeneCounts --twopassMode Basic
Counts per gene were calculated using featureCounts software. Parameters used: -s 2 -T 20 -t exon -g gene_id -a gencode.v28.chr_patch_hapl_scaff.annotation.gtf
Differential expression analysis used the DESeq2 software in R environment. Procedures included: Normalization of count values between samples using a geometric mean, estimation of sample-wise factors to correct for library size variability (for example), estimation of dispersion (i.e. variance, scatter) of gene-wise values between conditions, negative binomial linear model and Wald test to produce p-values, removal of low-expression outliers (using Cook's distance) results to optimize for p-value adjustment and finally, multiple testing adjustment of p-values with Benjamini-Hochberg procedure.
Genome_build: GRCh38.p12 reference genome (for processed data file included in the GSCs_RNAseq_1.xlsx) and GRCh38.p13 from GENCODE human release v.33 (for processed data file included in the GSCs_RNAseq_2.xlsx)
Supplementary_files_format_and_content: Raw count matrix of sequencing reads (featureCounts meta-feature level) as .xlsx

Submission date

Mar 23, 2021

Last update date

Mar 24, 2021

Contact name

Vadim Le Joncour

E-mail(s)

[email protected]

Phone

+358504486441

Organization name

University of Helsinki