GSM6215523
RNA was harvested using Rneasy mini plus kit (Qiagen). RNA sequencing libraries were prepared following the SMARTseq2 protocol. RNA was quantified using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific). Reverse transcription with the input material of 2ng was performed using SuperScrpit II (Invitrogen) in the presence of oligo-dT30VN, template-switching oligonucleotides and betaine. The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Roche), 100 nM ISPCR primer and 12 cycles of amplification. Following purification with Agencourt Ampure XP beads (1:1 ratio; Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer High Sensitvity DNA Kit (Agilent). The amplified cDNA (200 ng) was fragmented for 10 min at 55 ºC using Nextera XT (Illumina) and amplified for 12 cycles with indexed Nextera PCR primers. The library was purified twice with Agencourt Ampure XP beads (0.8:1 ratio) and quantified on a Bioanalyzer using a High Sensitvity DNA Kit. The libraries were sequenced on NovaSeq 6000 (Illumina) in paired-end with a read length of 2x51bp according to the manufacturer's protocol for dual indexing. Image analysis, base calling and quality scoring of the run are processed using the manufacturer's software Real Time Analysis (RTA 3.4.4) and followed by generation of FASTQ sequence files.