HeLa nucleoli PACBIO
Total RNA from isolated HeLa nucleoli was poly-adenylated by Poly(A) Polymerase Tailing Kit (Epicentre) and depleted of rRNA by the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen). RNA was then reverse transcribed by the SMARTer PCR cDNA Synthesis Kit (Clontech) and amplified for 15 cycles using KAPA HiFi PCR Kit (KAPA biosystems). cDNA was then separated into two fractions by size using 0.5X and 1X AMPure PB Beads (Pacific Scientific), respectively. SNUL-1 was then enriched from the two fractions by xGen capture procedure with SNUL-1 Probe 4 using the xGen hybridization and Wash Kit (IDT). Another round of PCR amplification was carried out after the capture. The two fractions were then combined. Library was prepared by Amplicon SMRTbell Prep (Pacific Scientific) and sequenced on LR SMRT cell with 20 h movie.