GSM8709841: COVID-2T_Patient_1_Memb; Homo sapiens; RNA-Seq
Tissue samples were manually pulverized on dry ice, transferred to pre-chilled tubes, and stored at -80°C. Nuclei isolations were performed with the 'hypotonic lysis buffer solution B' [Martin et al. 2023]. Up to 200 mg of tissue was added to 5 mL of 'hypotonic lysis buffer B' for 5 to 25 minutes on ice with periodic agitation. For samples under 50 mg, 500 uL of 'hypotonic lysis buffer B' was added directly to the 1.5 mL sample tubes and a RNase-free pestle was used for 6 to 12 strokes to further dissociate the tissue. Released nuclei were passed through a 70-micron cell strainer, pelleted (500xg, 5 minutes, 4°C), and resuspended in 1 mL of cold '0.3 M SPBSTM' (Martin et al. 2023). Pre-chilled fixative (5 mg of DSP resuspended in 100 uL DMSO per 4 mL of methanol) was incrementally added for every 1 mL of nuclei resuspended in '0.3 M SPBSTM' and fixed on ice for 5 to 10 minutes. The nuclei were rehydrated by gradually adding 10 mL of '0.3 M SPBSTM' for every 5 mL of fixed nuclei suspension prior to centrifugation (500xg, 5 minutes, 4°C) and resuspended in 1 mL of '0.3 M SPBSTM'. Nuclei suspensions processed in the third sci-RNA-seq3 experimental batch were additionally  overlaid on 7 mL of a 1.4 M sucrose buffer (1.4 M sucrose, 1X phosphate buffered saline (8 mM Na2HPO4 (pH 7.4), 2 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl), 3 mM MgCl2) and centrifuged at 3,000xg, 20 minutes, 4°C to remove excess debris; the pelleted nuclei were again resuspended in 1 mL of '0.3 M SPBSTM' and passed through a 20-micron cell strainer. After counting, nuclei aliquots were centrifuged (500xg, 5 minutes, 4°C), supernatant aspirated, and pelleted nuclei stored at -80°C until use. Methods for the first two sci-RNA-seq3 experimental batches largely followed those described by Cao et al. [Cao et al. 2019] with the following modifications: samples were counted with the ImageExpress Pico System (Molecular Devices), 2 μl of oligo(dT) primers were added to each well with 80,000 nuclei for reverse transcription, and tagmentation was performed using 1/40 μl per well of i7-loaded TDE1 enzyme prepared at the BAT-Lab following published protocols [Hennig et al. 2-18]. Libraries were sequenced on an Illumina Nextseq 200 cycles P3 kit with the following cycles: read 1, 34 cycles; read 2, 100 cycles; index 1, 10 cycles; index 2, 10 cycles. Methods for the third sci-RNA-seq3 experimental batch largely followed the optimized protocol described by Martin et al. (Martin et al. 2023) with the following modifications: samples were counted with the ImageExpress Pico System (Molecular Devices), 20,000 nuclei were loaded per reverse transcription well, tagmentation with N7 adaptor-loaded Tn5 transposases (Diagenode cat. no. C01070010-20) was performed at 1/80 uL per well across PCR plates, final library clean up was 0.5X AMPure XP supernatant followed by Qiagene GeneRead Size Selection purification column. The library was sequenced on an Illumina NovaSeq S4 flow cell at the University of Washington Northwest Genomics Center core sequencing facility with the following cycles: read 1, 34 cycles; read 2, 100 cycles; index 1, 10 cycles; index 2, 10 cycles. Read alignment and gene count matrix generation was performed using the Brotman Baty Institute (BBI) pipeline for sci-RNA-seq3 (https://github.com/bbi-lab/bbi-dmux; https://github.com/bbi-lab/bbi-sci). The human GRCh38 build and annotation from Ensembl (release 99) was used as the reference genome. The 3' UTR annotations of genes and transcripts were extended by 500 bp to avoid misclassifying genic reads as intergenic, and haplotype sequences were removed from the FASTA to avoid mapping inefficiencies due to multimapping. single-nuclei RNA libaries (sci-RNA-seq3)