GSM5693472: SPLiT-seq Mouse Brain; Mus musculus; RNA-Seq
Single nucleus suspensions were generated from n=48 (6 groups, 4 female 4 male) of tissue as previously described (Morabito et al., 2020) except after the 40μm filtration, nuclei were immediately processed using the Nuclei Fixation kit (Parse Biosciences, formerly Split Biosciences) and then cryopreserved with DMSO until library preparation. Nuclei isolation was performed in randomized groups of eight samples. library preparation was performed in a single batch of all 48 samples. Single-nucleus RNAseq libraries were generated with the Single Cell Whole Transcriptome library preparation kit (Parse Biosciences), which utilizes split-pool ligation-based transcriptome-sequencing (SNucseq)as described in in (cite Rosenberg 2018 Science). Briefly, each sample, normalized by nuclei number, was added to one well of a 96-well plate to perform reverse transcription, adding a well-specific barcode (first barcode). All the wells were then pooled together and randomly distributed across a new 96-well plate with new well-specific barcodes (second barcode) for ligation. After ligating the second barcode, the wells are again pooled and randomly distributed across another 96-well plate for a third round of barcoding. Throughout this process, the nuclei are still intact but permeabilized to allow the reactions to append three different barcodes to each transcript. The nuclei were then pooled into 8 sublibraries of 12,500 nuclei and lysed to allow the construction of the final cDNA libraries, in which a fourth, sublibrary-specific barcode is added. This thus results in single-nucleus transcriptomes that can be identified based up on the combination of the four different barcodes. cDNA library quality was assessed using the Agilent 4200 Tapestation, and concentrations were determined using Qubit. Libraries were sequenced using the Illumina NovaSeq 6000 S4 at UCI's Genomics High-Throughput Facility for a sequencing depth of 50,000 read pairs/cell. library preparation was performed in a single batch of all 48 samples. Single-nucleus RNAseq libraries were generated with the Single Cell Whole Transcriptome library preparation kit (Parse Biosciences), which utilizes split-pool ligation-based transcriptome-sequencing (SNucseq)as described in in (cite Rosenberg 2018 Science). Briefly, each sample, normalized by nuclei number, was added to one well of a 96-well plate to perform reverse transcription, adding a well-specific barcode (first barcode). All the wells were then pooled together and randomly distributed across a new 96-well plate with new well-specific barcodes (second barcode) for ligation. After ligating the second barcode, the wells are again pooled and randomly distributed across another 96-well plate for a third round of barcoding. Throughout this process, the nuclei are still intact but permeabilized to allow the reactions to append three different barcodes to each transcript. The nuclei were then pooled into 8 sublibraries of 12,500 nuclei and lysed to allow the construction of the final cDNA libraries, in which a fourth, sublibrary-specific barcode is added. This thus results in single-nucleus transcriptomes that can be identified based up on the combination of the four different barcodes. cDNA library quality was assessed using the Agilent 4200 Tapestation, and concentrations were determined using Qubit. Libraries were sequenced using the Illumina NovaSeq 6000 S4 at UCI's Genomics High-Throughput Facility for a sequencing depth of 50,000 read pairs/cell.