GSM8521943: Sample_ISM247626-2; Homo sapiens; OTHER
RNA was extracted from tissue using TRIzol (Invitrogen, Waltham MA) according to manufacturer's instructions, followed by addition of chloroform and centrifugation to separate the solution into aqueous and organic phases. Finally, RNA was precipitated, washed, solubilized and quantified via Bioanalyzer (Agilent, Santa Clara CA) where an RNA Integrity Number (RIN) score of 7 was set as the quality threshold. Starting with Total RNA, the library constructed by following PacBio protocol. Briefly, it starts with first-strand synthesis, then, pool the diluted first-strand reactions for large-scale amplification. Performing cycle number optimizations to minimize PCR bias following the PCR cycle optimization protocol. The determined cycle number is used to generate a sufficient amount of double-stranded cDNA product for SMRTbell library construction. AMPure methods (1X and 0.40X ) are used for purification, then pooled the fraction 1 (1X) and fraction 2 (0.40X) for SMRTbell template preparation. At least 1 μg of pooled cDNA for library construction. The steps include repair DNA damage, repair ends, prepare blunt ligation reaction, adding exonuclease to remove failed ligation products, and purifying SMRTbell templates. Finaly, anneal and bind non-size selected SMRTbell templates. A combined procedure to construct and pool a non-size selected Iso-Seq library with a size selected Iso-Seq library for sequencing on the Sequel System. Incorporating a size selection step using BluePippinTM enables one to increase the sequencing yield of >4 kb transcripts. Using the Clontech SMARTer PCR cDNA Synthesis Kit, RNA is synthesized to cDNA and subsequently amplified to generate double-stranded cDNA. One portion of the amplified cDNA product is used directly to construct a non-size selected SMRTbell library as summarized above. A second portion of the amplified cDNA product can be size-selected using either BluePippinTM, and then the size-fractionated cDNA is used to construct a size-selected SMRTbell library. Finally, the non-size selected SMRTbell library and the size-selected SMRTbell library are separately annealed and bound, and then pooled together for sequencing on the Sequel System.