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| Status |
Public on Dec 21, 2022 |
| Title |
CH22_RNA_DOX_SNF5pos_Rep2 |
| Sample type |
SRA |
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| Source name |
CH22
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| Organism |
Homo sapiens |
| Characteristics |
cell line: CH22
cancer type: chordoma
treatment: 72hr doxycycline (SN5 add-back)
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| Treatment protocol |
For all RNA-seq studies, 2x10^6 CH22- and UM-Chor5- pIND20+fSN5-HA cells were plated into 10cm dishes and induced with DOX (1 μg/μl) for 72 hours.
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| Growth protocol |
The CH22, UM-Chor5, and TTC642 cell lines were cultured in RPMI 1640 (#11875-093, Gibco, Grand Island, NY) and supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO). The pIND20+SNF5-HA cell lines were cultured in RPMI 1640, supplemented with 10% TET-FREE FBS (#100-800, GeminiBio, Sacramento, CA) and 400 ug/ml G418/Geneticin 50mg/ml (#10131-035, Gibco, Grand Island, NY). All cells were maintained in a humidified incubator at 37°C with 5% CO2. All were used within 10 passages of their initial arrival to minimize chances of cross-contamination and tested for mycoplasma contamination by DAPI staining and PCR for the detection of mycoplasma ribosomal DNA.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from frozen cell pellets in biological triplicate using Zymo Quick-RNA Miniprep Kit (Zymo cat# R1054).
Total RNA was prepared by Novogene Inc. using the Illumina TruSeq mRNA v2 kit with an input of 500ng of RNA for each sample to produce unstranded RNA libraries following the manufacturer’s protocol. Final RNA libraries were quantified using the Qubit High Sense Reagent kit and Agilent Tapestation HSD1000 tapes. Libraries were equimolar pooled and paired-end sequenced across three lanes of an Illumina HiSeq 4000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
CH22_SNF5pos.v.SNF5neg_all_genes.txt
chordoma_MRT_SNF5_addback_samples_RNA_combined_VST_expression.txt
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| Data processing |
Reads were trimmed using cutadapt (v1.12) using options -a GATCGGAAGAGC -A GATCGGAAGAGC and --minimum-length 36 to remove any sequencing adapters.
After trimming, reads were filtered for quality using the fastq_quality_filter in FASTX-Toolkit (v0.0.12), with options -Q 33, -p 90, and -q 20. Cell lines JHC7, MUG-Chor1, U-CH1, U-CH2, and UM-Chor1 were run with option -Q 64 instead.
Reads were aligned to the hg19 genome using STAR (v2.5.4b) with options --quantMode TranscriptomeSAM, --outFilterMismatchNmax 2, --alignIntronMax 1000000, --alignIntronMin 20, --chimSegmentMin 15, --chimJunctionOverhangMin 15, --outSAMtype BAM Unsorted, --outFilterType BySJout, and --outFilterMultimapNmax 1.
To calculate the RNA abundance values in transcripts per million (TPM), Salmon (v0.8.1) tool quant was used.
Samtools (v1.3.1), bedtools (v2.26), Python (v3.7.9), and R (v3.3.1) were used to interconvert files for downstream analyses.
DESeq2 (v1.14.1) was used to determine which genes had significantly differential RNA abundance.
PCA plots were generated using the plotPCA function within DESeq2 using all RNA abundance values (TPMs). gProfiler GOSt was used to identify Reactome pathways and p-values associated with differentially expressed genes. Heatmaps of gene TPMs were made using Morpheus. Boxplots were made in Python using seaborn boxplot and stripplot commands using the log2 fold changes between the mean TPMs across replicates between SMARCB1 positive and SMARCB1 negative samples. Upset plot was generated in R with the UpSetR package.
Assembly: hg19
Supplementary files format and content: DESeq2 files contain the DESeq2 analyses for every genes in the titled comparison.
Supplementary files format and content: Expression matrix is a tab-delimited file containing the VST-normalized gene expression for all genes and all samples analyzed in the manuscript.
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| Submission date |
Nov 15, 2022 |
| Last update date |
Dec 21, 2022 |
| Contact name |
Austin J Hepperla |
| E-mail(s) |
hepperla@unc.edu
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| Organization name |
University of North Carolina at Chapel Hill
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| Department |
Genetics
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| Street address |
7018B Mary Ellen Jones Building
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE217999 |
SMARCB1 Loss in Poorly-differentiated Chordomas Drives Tumor Progression |
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| Relations |
| BioSample |
SAMN31737635 |
| SRA |
SRX18272236 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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