GSM9321044: Control_RNAseq_rep3; Homo sapiens; RNA-Seq
Total RNA was isolated from fresh frozen specimens with TRIzol reagent according to the manufacturer's instructions (Invitrogen). Samples in OCT were cut in 20 µm-thick sections and 20-30 sections per specimen were used for extraction with TRIzol (Invitrogen) according to manufacturer's instructions. The cDNA libraries were first constructed using SMART-Seq v4 Ultra low input RNA Kit (Clontech Laboratories, Takara Bio Company; Mountain View, CA, USA) according to manufacturer's instructions. Briefly, the first-strand cDNA was synthesized using total RNA from the tissue by the 3' SMART-Seq CDS Primer II A and template switching was performed by SMART-Seq v4 Oligonucleotide at the 5' end of the transcript.  Then, cDNA from SMART sequences was amplified by PCR Primer II A. After 17 cycles of long-distance PCR, amplified cDNA was purified using the Agencourt AMPure XP Kit (Beckman Coulter, Cat.No. A63882). The quality of cDNA construction was validated using Agilent Tapestation 2200 system. Subsequently, 150pg of amplified cDNA was used for Illumina library preparation using Nextera XT DNA Library Preparation Kits (Illumina Inc., San Diego, CA, USA). Libraries with unique index were pooled together in equimolar ratio and sequenced for paired-end 100pb sequencing on an Illumina Novaseq 6000 system.