Sequencing of mice brains

Sequencing of brains dissected from five male C57BL6/J wild-type mice sacrificed by cervical dislocation at 3 months of age. Brain tissues were homogenized using FastPrep and total RNA extracted using the 16 LEV simplyRNA Purification Kit for Maxwell. Samples were spiked-in using SIRV subset E0. For PacBio IsoSeq, cDNA synthesis and sample barcoding were performed using the NEBNext Single Cell/Low Input cDNA synthesis amplification kit, and four libraries were generated using IsoSeq SMRTbell prep kit 3.0. Eight SMRT Cells were sequenced on a PacBio Sequel IIe. For nanopore samples, cDNA synthesis and sample barcoding were performed using the Nanopore SQK-PCB111-24 PCR-cDNA Barcoding Kit, following the standard protocol and generating 8 libraries per sample. The 8 libraries were then mixed and split into 8 aliquots. Eight rounds of sequencing were conducted on R9 flow cells (FLO-PRO002) using a Nanopore PromethION 2 . For PacBio Kinnex libraries, RNA from 3 mice brains were used for library preparation using Kinnex full-length RNA kit and sequenced on a Revio at Pacific Biosciences.