GSM7663060: striatum_YAC128_AON_A1906953; Mus musculus; RNA-Seq
RNA was isolated from striatum using the PureLink RNA mini kit (Thermo Fisher Scientific) including a 15 minute DNase treatment step. Tissue was homogenized in 700 µl lysis buffer in MagNA Lyser Green beads tubes (Roche) with bullet blender Storm 24 (Next Advance) for 2-3 minutes at setting 8. RNA was eluted in 30 µl nuclease-free water and concentrations were measured using the Nanodrop (Thermo Fisher Scientific). Full-length cDNA generation from RNA-seq data was performed using the mcSCRB-seq method with modifications (https://doi.org/10.1038/s41467-018-05347-6). Instead of single cells, 100 ng of isolated total RNA was used. Polyvinylpyrrolidone (PVP) was used instead of PEG8000 to reduce reaction viscosity. 0.5% PVP gave similar results as 7.5% PEG8000. The oligodT primer was replaced with a primer designed for scifi-RNA-seq (https://www.nature.com/articles/s41592-021-01153-z) containing partially Read 2 sequence primer from the Illumina platform, the UMI, and the well/sample barcode (example: SCIFI_LIG384_001 GAGTTCAGACGTGTGCTCTTCCGATCT-NNNNNNNN-AAGTGATTAGCAA-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN). Samples were uniquely barcoded using the well barcode, and the handle for Read 2 sequence primer allowed further barcoding of a pool of samples by adding the i7 Illumina barcode and the p7 sequence (CAAGCAGAAGACGGCATACGAGAT[i7_barcode]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT). The pooled cDNA libraries were generated using the 2x Kapa HiFi HotStart mix (Roche). The Illumina library prep was performed using the Kapa Hyper Plus kit (Roche) with fragmentation, end-repair, A-tailing, and double SPRI purification. Custom indexed adapter barcodes were used to barcode the pool of samples using the i5 Illumina barcode and introduce the p5 sequence to complete the Illumina library (top_adapter: AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATC*T, bot_adapter GATCGGAAGAGCGT/3InvdT/). The ligated products were purified using Ampure beads, and PCR with Kapa Mastermix (Roche) and p5 and p7 primers was performed for enrichment. After double SPRI size selection, the Illumina library was quantified and checked for size distribution. Illumina paired-end sequencing was performed on a NovaSeq 6000 using the manufacturer's instructions.  After sequencing, Read 1 contained the cDNA information, and Read 2 contained only the UMI. To facilitate pipeline analysis, the UMI from the Read 2 fastq file was added to the beginning of the same read ID in the Read 1 fastq file, resulting in a modified Read 1 fastq file starting with the UMI followed by the cDNA sequence, which was used in the RNA-seq pipeline.