title: GSM8497706: N1_PAIsoseq; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted by TRIzol reagent (Invitrogen, #15596026) and chloroform (Sinopharm, #10006818), and precipitated with isopropanol (Sinopharm, #80109218) and dissolved with nuclease-free water. Purified total RNA were used for templated end extension by dU-containing end-extension oligo and Klenow fragment 3'-5' exo- (NEB, #M0212L). Then the templated RNA were cleaned by USER enzyme (NEB, #M5505S) and RNA Clean & Concentrator-5 kit (Zymo Research, #R1016). Purified templated RNA were retro-transcripted into cDNA by using SuperScript Ⅱ reverse transcriptase (Invitrogen, #18064-014) and TSO primer[1]. The full-length cDNA were further pre-amplified by using 2×KAPA HiFi HotStart ReadyMix (KAPA Biosystems, #KK2601) and cleaned by SPRIselect beads (Beckman Coulter, #B23318). For each sample, 20 ng purified cDNA were used for final amplification and cleaning as pre-amplification. Finally, cDNAs from different samples were mixed together according to the concentration and fragments length, and 500ng mixed cDNA were used for SMRTbell template library and sequencing by using Pacbio sequell Ⅱ.