|Public on May 30, 2017
|HCC1143 cell line
|treatment: 1uM BEZ235
cell line: HCC1143
|Two plates of each line were then treated with 1μM Trametinib, two plates with 1μM BEZ235, two plates with 1μM of both agents, and two plates with 0.05% DMSO as a control. Plates were incubated for 72hr, then total RNA was isolated using the QIAGEN RNeasy mini kit according to manufacturers instructions.
|HCC1143 cells were cultured in RPMI 1640 + 10% fetal bovine serum and 10g/ml penn/strep, cells were maintained at 37°C in a 5% CO2 atmosphere.
|Total RNA was isolated using the QIAGEN Rneasy mini kit according to manufacturers protocol
Libraries were made with the Illumina Truseq Samples Prep v2 kit with 150ng RNA using manufacturer's protocol.
Poly A capture: total RNA was run on the Bioanalyzer (Agilent) to verify intactness. Following this test, RNA-seq libraries were constructed using the TruSeq RNA sample prep protocol (Illumina). Poly(A)+ RNA was isolated using oligo-dT bound to magnetic beads. Poly(A)+ RNA was then chemically fragmented. RNA fragments were converted to double stranded cDNA using random hexamer priming. Fragment ends were treated to remove overhanging nucleotides and then a single “A” was added to the 3’ end of each strand to facilitate ligation. Illumina adaptors with barcode sequences was ligated to the fragments. The resulting libraries were then amplified using polymerase chain reaction with a limited number of rounds. Unincorporated nucleotides and adaptor dimers were removed using AMPure XP beads (Agencourt). Libraries were combined for multiplexing and sequenced on the NextSeq 500
|Illumina NextSeq 500
|PI3K/mTOR inhibitor treated HCC1143.
|Base calling - using Illumina RTA version 2.4.11
Demultiplexing was done with Bcl2fastq 18.104.22.168
Reads were trimmed to 44 bases (first 4 bases were discared, next 44 were kept, and remaining bases were also discarded).
Trimmed reads were aligned to hg19 with Bowtie version 1.0.0 (Up to 3 mismatches were allowed, but unique best matches were required.)
R scripts were used to count tags that aligned to the exons of UCSC RefSeq gene models and calculate RPKM values.
Supplementary_files_format_and_content: Supplementary files are tab delimited text files with tag counts and RPKM values for each RefSeq gene model.
|May 17, 2017
|Last update date
|May 15, 2019
|Oregon Health & Science University
|Molecular Medical Genetics
|Dr. Rosalie Sears
|2730 SW Moody Ave
|Human basal-like breast cancer cell line HCC1143 treated with BET inhibitor JQ1 in combination with MEK inhibitor Trametinib or PI3K/mTOR inhibitor BEZ235