GSM6752856
RNA was harvested using an RNeasy mini kit (Qiagen). 50 ng of total RNA was used for the construction of sequencing libraries. RNA libraries were prepared using bulk RNA barcoding and sequencing (BRB-Seq) with some modifications. Reverse transcription (RT) was performed with 50 ng total RNA per sample using SuperScript IV (Invitrogen) and a distinctly barcoded oligo(dT)30 primer for each sample (modified with the Illumina TruSeq Read 1 priming site instead of Nextera Read 1) with the following thermocycling program: 50 °C, 15 min; 55 °C, 15 min; 60 °C, 15 min; 80 °C, 15 min. After RT, up to 24 barcoded first-strand cDNA samples were pooled into a single tube. For each cDNA pool, second-strand cDNA was synthesized by the Gubler-Hoffman nick translation approach per the BRB-Seq protocol. Then, instead of Tn5 tagmentation, each cDNA pool was fragmented and end-repaired with the NEBNext Ultra II FS DNA Module (NEB) at 37 °C for 6 min, followed by 65 °C for 30 min. 37.5 pmol DNA adaptor containing Illumina Read 2 was duplexed and ligated using optimized ligation conditions at 20 °C for 2 h before purifying ligation products with sequential 1.0X AMPure XP SPRI cleanups (Beckman Coulter). Each cDNA pool was amplified with 12 cycles of PCR using a P5 containing primer and a distinct multiplexed i7 indexing primer (Chromium i7 Multiplex Kit, 10X Genomics, Pleasanton, CA), size-selected with 0.55/0.75X double-sided size-selection with SPRIselect (Beckman Coulter), then amplified for another 6 cycles of PCR using P5 and P7 primers, before a final 0.7X SPRIselect cleanup.