Poly(A) inclusive RNA isoform sequencing (PAIso-seq) of mouse bulk GV oocytes
Total RNA from GV stage oocytes are used to end extended with dNTP and Klenow fragment (exo-, NEB) on a dU-containing DNA template at 37 for 1 hour. dUs were digested by USER enzymes (NEB) at 37 for 30 minutes. End extended RNA (>200 nt) were cleaned up and concentrated with RNA Clean & Concentrator-5 kit (Zymo Research): the sample was mixed thoroughly with 50 l RNA Binding buffer and 50 l 100% ethanol; the mixture was transferred into a Zymo-Spin IC Column and centrifuge; after washing, the nucleic acids was eluted by adding 6-8 l nuclease-free water directly to the column matrix and centrifuging. Prepared RNA was stored at -80 or used immediately. The cleaned End extended RNA was reverse transcribed with template switching similarly to Smart-seq2 method with RT primer and TSO. A total of 28 PCR cycles of amplification was performed using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with PCR primers. Amplified cDNA products were size selected by Pure PB beads and BluePippin, made into Iso-Seq SMRTbell Template libraries (SMRTbell Template Prep Kit). The libraries were annealed with the sequencing primer and bind to polymerase, finally the polymerase-bound template was bound to Magbeads and sequenced using a PacBio Sequel instrument.