GSM8068591: CTC35; Homo sapiens; RNA-Seq
Healthy control PBMCs were isolated using Ficoll-Paque Density Gradient Separation. CTCs were enriched using the StemCell Technologies EasySep Direct Human CTC Enrichment Kit. Single-cell RNA-seq libraries were prepared following the Smart-seq2 protocol from Picelli et al. 2014. Some subtle differences in library prep thermocycler conditions were required to optimize the preps for patient-derived CTCs when compared to A375s or the “mini-bulk” positive control reactions, as per Smart-seq2's recommendations. For single A375s, patient-derived CTCs, and PBMCs, samples were preamplified (Smart-seq2 step 14) for 18 cycles, corresponding to the expected 10 pg of input RNA from the initial cell lysis. “Mini-bulk” positive control preps were preamplified for 16 cycles, corresponding to the 100 pg of input RNA. After bead purification, library concentration was determined with a Qubit dsDNA HS Assay Kit (Thermofisher cat. no. Q32851, Waltham, MA, USA) and an Agilent Bioanalyzer High Sensitivity DNA kit (cat. no. 5067-4626, Santa Clara, CA, USA). Next, 1 ng of bead-purified, preamplified dsDNA library was loaded into the tagmentation reaction at step 28 of the Smart-seq2 protocol. The final amplification reaction at step 33 was carried out for 12 cycles. All steps not discussed here were carried out as described in Picelli et al. 2014.