title: GSM8072862: Primary mouse astrocytes long-read rep2 [AC2]; Mus musculus; OTHER
library_construction_protocol: RNA was extracted from sEVs using the Qiagen RNeasy Micro kit according to the manufacturer's instructions, including an on-column DNase treatment. RNA was also extracted from sEV-source cells using the Qiagen RNeasy Mini kit according to manufacturer's instructions including on-column DNase treatment. cDNA libraries from sEV RNA and cell RNA were generated using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® underwent 10 cycles of reamplification using the same kit. Samples were cleaned up with 1.2X ProNex beads (VWR NG2001). 20ng of cDNA from each human sample or 100ng of each murine sample was used as input for the preparation of SMRTbell® libraries according to the “PacBio® Barcoded Overhang Adapters for Multiplexing Amplicons” protocol using the SMRTbell express template prep kit 2.0. Human samples were pooled according to their group: AD bulk, AD sEVs, ND bulk, or ND sEVs. Sequencing primer v4 kit and Sequel II Binding Kit 2.1 were used for human samples. Each human sample group was sequenced on 1 SMRTCell on the PacBio Sequel II with a movie time of 10 hours. Murine samples were combined by sample type: the first pool consisted of astrocyte, microglia, and neurons cells and the second pool consisted of astrocyte, microglia, or neuron sEVs. Sequencing primer 3.1 kit and Sequel II Binding Kit 3.1 were used for murine samples. Each pool was sequenced on 2 SMRTCells on the PacBio Sequel II with a movie time of 24 hours.