cuSCC Dataset C
Poly(A)+ RNA was isolated from 3 µg of total RNA using the Oligotex Mini kit (Qiagen) according to manufacturer’s instructions. The resulting poly(A) RNA was then amplified using the MessageAmp II aRNA Amplification Kit (Ambion, Austin, TX) using an in vitro transcription time of 14 hours at 37°C to generate aRNA.  200ng of aRNA was used to generate libraries for transcriptome sequencing using an adaptation of the protocol previously described by Yozwiak et al.(Yozwiak et al.).  In order to multiplex up to 16 samples within 1 sequencing library, aRNA samples were randomly primed and reverse transcribed using a primer containing a 14-bp sequence common to the 3’end of both Illumina adapters, a random monomer followed by a unique 3-bp barcode, and a random hexamer (pr1A_barcode). Second-strand synthesis was primed using pr1A followed by PCR amplification using the 18-bp Illumina/barcode sequence without the hexamer (pr1B_barcode) for 25 cycles. PCR products were purified using DNA Clean and Concentrator columns (Zymo Research, Orange, CA). 200ng of each individually barcoded sample were mixed together to generate a library of up to 16 samples, with each sample marked by a unique 3-bp barcode.  Library purification, size selection, and amplification proceeded as previously described(Yozwiak et al.).