DGE-seq Gene expression data for 7 breast cancer cell lines treated with abemaciclib, palbociclib, ribociclib at 0.1, 0.3, 1, or 3 μM, or alvocidib at 0.1 or 1 μM for 6 hours - Dataset (ID:20373)
|HMS Dataset ID:||20373|
|Dataset Title:||DGE-seq Gene expression data for 7 breast cancer cell lines treated with abemaciclib, palbociclib, ribociclib at 0.1, 0.3, 1, or 3 μM, or alvocidib at 0.1 or 1 μM for 6 hours|
|Project Summary Page(s):||Publication Summary|
|Screening Lab Investigator:||Caitlin Mills|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||We collected mRNA-seq data using the high-throughput, low-cost RNA sequencing method 3’ Digital Gene Expression (DGE-seq) for seven breast cancer cell lines that were exposed for 6 hours to palbociclib, ribociclib, or abemaciclib at four concentrations or to alvocidib at 2 concentrations. Log2 fold change data are provided|
1) Cells were plated at densities ranging from 500 to 2000 cells per well in a 384-well Cell Carrier plate (Perkin Elmer, Waltham, MA) and allowed to adhere for 24 hours.|
2) Cells were treated with the CDK4/6 inhibitors, alvocidib, or DMSO using a D300 Digital Dispenser (Hewlett-Packard, Palo Alto, CA).
3) After six hours, the cells were washed once with PBS using an EL405x plate washer (BioTek, Winooski, VT), 10 µl of 1X TCL lysis buffer with 1% (v/v) ß-mercaptoethanol (Qiagen, Hilden, Germany) was added per well, and the plates were stored at -80˚C until the RNA extraction was performed.
4) For RNA extraction, the cell lysate plate was thawed, vortexed briefly, and centrifuged for 1 min at 1000 rpm. Using a BRAVO (Agilent, Santa Clara, CA) liquid handler, the lysate was mixed thoroughly before transferring 10 µl to a 384 well PCR plate. 28 µl of SPRI beads (Beckman Coulter Genomics, Chaska, MN) were added directly to the lysate, mixed and incubated for 5 min. The plate was transferred to a magnetic rack to aggregate the beads, and incubated for 5 min prior to removing the liquid. The beads were washed with 80% ethanol twice, allowed to dry for 1 min, 20 µl of nuclease free water was added per well, the plate was removed from the magnetic rack and the beads were thoroughly resuspended. Following a 5 min incubation, the plate was returned to the magnetic rack and incubated an additional 5 min before transferring the supernatant to a fresh PCR plate.
5) 5 µl of the supernatant was transferred to a separate plate containing RT master mix and 3’ and 5’ adapters for reverse transcription and template switching (Soumillon et al., 2014), and incubated for 90 min at 42˚C. The cDNA was pooled and purified with a QIAquick PCR purification kit according to the manufacturer’s directions with the final elution in 24 µl of nuclease free water. This was followed by an exonuclease I treatment for 30 min at 37˚C that was stopped with a 20 min incubation at 80˚C.
6) The cDNA was then amplified using the Advantage 2 PCR Enzyme System (Takara, Fremont, CA) for 5 cycles, and purified using AMPure XP magnetic beads (Beckman Coulter Genomics, Chaska, MN). Library preparation was completed with 55 ng input using a Nextera DNA kit (Illumina, San Diego, CA) following the manufacturer’s instructions, amplified 5 cycles, and purified with AMPure XP magnetic beads (Beckman Coulter Genomics, Chaska, MN).
7) A Pippin PREP purification of the sample from 300-800bp was performed, it was then quantified by qPCR and sequenced on a single Illumina NextSeq run with 75bp paired end reads at the Harvard University Bauer Core Facility.
8) Reads were processed to counts through the bcbio-nextgen single cell/DGE RNA-seq analysis pipeline (bcbio-nextgen.readthedocs.io/en/latest/contents/pipelines); a brief description follows: The well barcode and UMIs were identified for all reads and all reads not within one edit distance of a known well barcode were discarded. Each surviving read was quasialigned to the transcriptome (GRCh38) using RapMap (Srivastava et al., 2016). Reads per well were counted using UMIs (Svensson et al., 2017), discarding duplicated UMIs, weighting multimapped reads by the number of transcripts they aligned to and collapsing counts to genes by adding all counts for each transcript of a gene.
|Assay Protocol Reference:||Soumillon, M., Cacchiarelli, D., Semrau, S., van Oudenaarden, A., and Mikkelsen, T.S. (2014). Characterization of directed differentiation by high-throughput single-cell RNA-Seq. BioRxiv.|
|HMS Dataset Type:||RNA-Seq|
|Date Publicly Available:||2020-06-24|
|Most Recent Update:||2020-06-24|