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Sample GSM6019828

Query DataSets for GSM6019828

Status

Public on Apr 08, 2022

Title

Sample166 rat GH3 RNA

Sample type

SRA

Source name

GH3 cells

Organism

Rattus norvegicus

Characteristics

tumor functional type: NA
therapy: Oct_8h

Treatment protocol

To obtain PitNET tissue-derived free-floating spheres, cells were grown in DMEM-F12 (Thermo Fisher Scientific, USA), containing 1x penicillin/streptomycin solution, 20 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich, Germany), 10 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich, Germany), and 1x B27 supplement (GIBCO, USA). Cell culture cultivation and incubation was performed at 37°C, 95% air, and 5% CO2. For the gene expression experiments, pituispheres were grown on a 6-well plate and treated with 10 μM octreotide or 10 μM cabergoline for 4, 8, 24, 48, and 72 h.
The GH3 cells were maintained in F12-K medium containing 15% horse serum, 2.5% FBS and 1x penicillin/streptomycin solution. For studying the effects of PitNET drugs on gene expression, the cells were incubated for 4, 8, 24, 48, and 72 h with 10 μM octreotide and 10 μM cabergoline. Cell culture cultivation and incubation was performed at 37°C, 95% air, and 5% CO2.
To obtain MSC cells were grown in DMEM-F12, supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, USA), 1% ITS (Corning, USA), and 100 µg/ml primocin (InvivoGen, USA) until confluent, then propagated and passaged 2–6 times. Cell culture cultivation and incubation was performed at 37°C, 95% air, and 5% CO2.

Growth protocol

Within 12 hours after surgery, PitNET tissue samples were processed for propagation. Tissue material was mechanically sliced into small pieces and washed in DMEM with 1x Antibiotic-Antimycotic solution (Thermo Fisher Scientific, USA). Enzymatic dissociation using Accutase solution (Thermo Fisher Scientific, USA) was carried out on a rotating platform for 20 min at 37°C in a humidified environment with 5% CO2. The cells were centrifuged for 5 min at 360 x g after the incubation period to obtain cell pellets. The cell pellet was treated with a red blood cell lysis solution (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) for 10 min to reduce contamination with red blood cells. To remove red blood cell debris, the sample was centrifuged and the cell pellet was washed twice. The obtained pellet was split into two portions. To obtain PitNET tissue-derived free-floating spheres, cells were grown in DMEM-F12 (Thermo Fisher Scientific, USA), containing 1x penicillin/streptomycin solution, 20 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich, Germany), 10 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich, Germany), and 1x B27 supplement (GIBCO, USA). To obtain MSC, culture cells were grown in DMEM-F12, supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, USA), 1% ITS (Corning, USA), and 100 µg/ml primocin (InvivoGen, USA) until confluent, then propagated and passaged 2–6 times. All cell culture cultivations and incubations were performed at 37°C, 95% air, and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA for transcriptome sequencing was extracted using AllPrep DNA/RNA/miRNA universal kit (Qiagen, Germany) according to manufacturer’s instructions from tumor tissue samples stored in RNAlater Tissue Storage Reagent (Sigma-Aldrich, USA). The concentrations of extracted RNA were measured using Qubit 2.0 with Qubit RNA HS kit (Thermo Fisher, USA). The quality of extracted RNA was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Following this the RIN and DV200 values were calculated to calculate the RNA input amounts for RNA-seq compatible library preparation.
Prior to the library preparation rRNA removal was performed using MGIEasy rRNA Depletion kit (MGI, China). After the rRNA depletion reverse transcription, second strand synthesis, and cDNA library preparation for NGS were carried out using MGIEasy RNA Directional Library Prep Set (MGI, China). The libraries were sequenced on DNBSEQ-G400 platform (MGI, China) with 35 M reads per sample and with 2 x 150 bp read length.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-G400

Description

Sample166

Data processing

Paired end data was trimmed with fastp(v0.23.2) software to retain reads with average quality of at least 20 (Phred score) and minimum read length of 100 base pairs.
Ribosomal RNA (rRNA) read alignment and removal was performed with SortMeRNA.
Quality and rRNA filtered reads were further quasi-mapped and quantified using Salmon(v1.6.0) against the GENCODE(v38) Homo sapiens genome for tissue and pituisphere samples while simultaneously performing GC bias, sequence level bias and positional bias correction to increase the number of high quality mappings. Tissue samples with quasi-mapping rate of at least 45% were selected for differential expression analysis.
Quality and rRNA filtered reads were further quasi-mapped and quantified using Salmon(v1.6.0) against Ensembl(v105) Rattus Norvegicus genome for the GH3 cell lines while simultaneously performing GC bias, sequence level bias and positional bias correction to increase the number of high quality mappings.
The quantified transcripts were imported in the R software and summarized to gene level counts using the tximeta package. Differential expression analysis was performed by DESeq2. Quantified read counts were filtered by frequency setting the count threshold at 10 and the sample frequency threshold as 33% of the smallest comparison group contrast size. Parametric dispersion fit type was chosen to model the counts, after also checking the fit of mean and local fits. Euclidean distances between samples and PCA graphs of 500 most variable genes were used to check for unforeseen variability within the comparison groups and possible batch effects. Subsequently Wald test was performed to determine expression differences both in PitNET type and SSA/DA therapy contrasts. The default independent filtering function was replaced with independent hypotheses weighing from the IHW package. Adjustment p values based on the mean expression level of each gene which was followed with multiple testing correction using the Benjamini-Hochberg adjustment.
Assembly: GENCODE v38 GRCh38
Assembly: Ensembl 105 mRatBN7.2
Supplementary files format and content: raw counts

Submission date

Apr 05, 2022

Last update date

Apr 08, 2022

Contact name

Raitis Peculis

Organization name

Latvian Biomedical Research and Study Centre

Street address

Ratsupites str 1 k-1

City

Riga