|
| Status |
Public on Apr 12, 2015 |
| Title |
CAL33B rep3 |
| Sample type |
SRA |
| |
|
| Source name |
CAL33
|
| Organism |
Homo sapiens |
| Characteristics |
resistant: Yes
|
| Treatment protocol |
Parental and acquired resistant were treated for 24 hours with 1uM BYL719 or DMSO.
|
| Growth protocol |
KYSE180 cultured in RPMI 10%FCS, CAL33 cultured in DMEM-F12 10%FCS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by Rneasy mini kit (Qiagen). After ribogreen quantification and quality control of Agilent BioAnalyzer(RIN>7), poly(A) RNA was isolated using Dynabeads® mRNA DIRECT™ Micro Kit(Life Technologies)from 1ug of total RNA. mRNA was then fragmentated using RNaseIII and purified . The Fragmented Samples quality and yield were evaluated using Agilent BioAnalyzer.
Fragmented material underwent Whole transcriptome Library preparation according to the Ion Total RNA-Seq Kit v2 protocol(Life Technologies), with 16 cycles of PCR. Samples were barcoded, template-positive Ion PI™ Ion Sphere™ Particles (ISPs) were prepared using the ion one touch system II and Ion PI™Template OT2 200kit v2 Kit (Life Technologies). Enriched particles were sequenced on a Proton sequencing system using 200bp version 2 chemistry. An average of 70 to 80 million reads per sample were generated.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
s_6_CAL33B_3
|
| Data processing |
Base calls done with Torrent Suite Software Basecaller
We performed differential expression analysis looking for genes that varied significantly between parental and resistant cell lines: CAL33 and KYSE180. The analysis was done jointly resulting in genes that largely agreed on fold change in both cell types.
For gene expression quantifications, first we converted the BAM files to FASTQ format. Reads were then quality trimmed using the fastx toolkit to remove bases that had a base quality score less than 10 and then reads shorter than 50bp were discarded. We did a two pass mapping procedure that first mapped with rnaStar,that maps reads genomically and resolves reads across splice junctions. Any reads that did not map in this pass were re-mapped using BWA MEM method. We merged the output BAM files from these two steps and then counted gene level expression with htseq-count (-s yes -m intersection-strict) and exon level expression was quantitate with DEXSeq. For the analysis we considered only coding genes.
After quantification of gene expression we assessed replicate quality in the different cell types and conditions and removed replicates that showed considerable amount of variation in gene expression ('CAL33_2', 'KYSE180B_1', 'KYSE180_1').
For the differential analysis we used the DESeq2 package in R. DESeq2 performs differential analysis using raw read counts, under a Negative Binomial noise model. Our linear model explained gene expression by cell-line type and whether it is parental or resistant off-drug.
For every gene G we fitted the model and tested whether was statistically significant, ie. was there a statistically significant change between parental and resistant cell lines. Model fitting was followed by FDR (BH) correction for multiple hypothesis testing.
Genome_build: hg19
Supplementary_files_format_and_content: Tab delimited text file with raw gene counts. Genes are labeled with ENSEMBL Gene ID Symbols
|
| |
|
| Submission date |
Sep 17, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas D Socci |
| E-mail(s) |
soccin@mskcc.org
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Department |
Computational & Systems Biology Program
|
| Lab |
Bioinformatics Core
|
| Street address |
1275 York Ave
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL17303 |
| Series (1) |
| GSE61515 |
AXL mediates resistance to PI3Kα inhibition by activating the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas. |
|
| Relations |
| BioSample |
SAMN03072688 |
| SRA |
SRX703444 |
