Sequencing of mice brains. 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.

对5只3月龄雄性C57BL6/J野生型（wild-type）小鼠经颈椎脱位法处死后剥离的脑组织开展测序分析。脑组织采用FastPrep进行匀浆处理，总RNA提取采用适配Maxwell系统的16 LEV simplyRNA纯化试剂盒，所有样本均加入SIRV子集E0作为spike-in对照（spike-in）。在PacBio IsoSeq测序流程中，采用NEBNext单细胞/低起始量cDNA合成与扩增试剂盒完成cDNA合成及样本条码标记，使用IsoSeq SMRTbell prep kit 3.0构建4个测序文库，随后在PacBio Sequel IIe测序平台上完成8个SMRT Cell的测序。针对纳米孔测序样本，采用Nanopore SQK-PCB111-24 PCR-cDNA Barcoding Kit，遵循标准操作流程完成cDNA合成与样本条码标记，每份样本构建8个测序文库；随后将8个文库混合并均分为8份等分试样，使用Nanopore PromethION 2测序平台搭配R9流动池（FLO-PRO002）开展8轮测序。针对PacBio Kinnex建库文库，以3只小鼠的脑组织总RNA为起始材料，采用Kinnex full-length RNA kit完成文库制备，并在Pacific Biosciences的Revio测序平台上完成测序。