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Sample GSM6234814

Query DataSets for GSM6234814

Status

Public on Jun 13, 2022

Title

H82-Control-Replicate 3

Sample type

SRA

Source name

lung

Organism

Homo sapiens

Characteristics

tissue: lung
cell line: H82
cell type: small cell lung cancer
genotype: WT
treatment: PBS-7 days

Treatment protocol

Treatment was performed with LSD1 inhibitor ORY-1001 (1𝝁M) or vehicle for 10 days

Growth protocol

H82 and H211 cell lines were grown in RPMI media supplemented with 10% FBS and 1% P/S. Cells were passaged every 3 days, STR-verified and routinely tested for mycoplasma.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany).
RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on 2 flowcell lanes. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

H82-Ctrl-3

Data processing

After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison. A gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF
Transcript counts and abundances were quantified from RNA-seq reads using Salmon v1.1.0 (Patro, R, et al 2017). RNA-seq raw reads were mapped to 25-mer indexed hg38 genome by Salmon. With default settings, mapping validation (--validatemappings), bootstrapping with 30 re-samplings (--numBootstraps), sequence specific biases correction (--seqBias), coverage biases correction (--posBias) and GC biases correction (--gcBias) were also enabled. Transcript to gene mapping based on Ensembl 92 (Zerbino, DR, et al 2018), normalized by size factor at gene level, and differential gene expression analyses were processed on Salmon output files using Sleuth v0.30.0 in gene mode (Subramanian, A, et al 2015). Differentially expressed genes were identified using the Wald test. Genes were marked as significant if the False Discovery Rates, q, calculated using the Benjamini-Hochberg method, was less than 0.05, and beta (Sleuth-based estimation of log2 fold change) > 0.58, which approximately correlated to a log2 fold change of 1.5 in our data.
Gene set enrichment analysis (GSEA) was performed on full sets of differential gene expression results on the previously mentioned comparisons. Genes were ranked on p value scores computed as -log10(p value)*(sign of beta). Gene set annotations include all gene sets deposited in the Molecular Signatures Database (MSigDB v7.0.1) (Subramanian, A, et al 2005; Liberzon, A, et al 2011). The significance level of enrichment was evaluated using permutation test and the p value was adjusted by Benjamini-Hochberg procedure. Any enriched gene sets with adjusted p value ≤ 0.05 were regarded as significant. This analyses were executed with the R package ClusterProfiler v3.18.1 (Yu, G, et al 2012).
Pathways and gene sets of interest were procured from GSEA-msigdb database (https://www.gsea-msigdb.org/gsea/index.jsp) and Gene Ontology resources (http://geneontology.org/). Interested pathways were highlighted in H82 and H211 GSEA dot plots. The log of the normalized TPM values for significant genes within the interested pathways, were rescaled using a z-score transformation, and plotted in a heatmap using the ComplexHeatmap v2.7.10 (Gu, Z, et al, 2016) Library in R 4.0.4.
Assembly: hg38
Supplementary files format and content: GEO_Sleuth_TPM.txt

Submission date

Jun 10, 2022

Last update date

Jun 13, 2022

Contact name

Bic MSKCC

Organization name

Memorial SLoan-Kettering Cancer Center

Street address

1275 York Ave.

City

New York