single-cell RNA-seq of mus musculus: adult lung
Murine lung samples were minced into 0.5 mm2 with a razor blade and digested with Liberase solution [RPMI-1640 (Nacalai Tesque) supplemented with 10% FBS, 10 mM HEPES pH7.2-7.4, 0.25 mg/ml Liberase TM (Roche), and 2 kU/mL DNase I (Merck)] at 37 C for 60 minutes. The cell suspension was agitated 20 times with an 18G needle (Terumo, Tokyo, Japan) after 20 minutes incubation, agitated 20 times with 21G needle (Terumo) after 40 minutes incubation, and agitated 10 times with 200 L pipette tip.Cell suspensions were passed through a 70 m cell strainer (BD Biosciences), centrifuged at 4C for 500 g for 5 minutes, and their supernatant was discarded. Cells were resuspended with 25% Percoll PLUS (Cytiva), agitated with an 18G needle five times. After under layered 65% Percoll PLUS, cell suspensions were centrifuged at 20C for 1,000 g for 20 minutes, and the middle layer was collected. Resultant cell suspensions were diluted thrice with preparation medium [RPMI-1640 supplemented with 5% FBS and 10 mM HEPES], centrifuged 500 g at 4C for 7 minutes, and their supernatants were discarded. Resultant cells were resuspended with 500 L of preparation medium[DMEM (Nacalai Tesque) supplemented with 5% FBS, 10 mM HEPES pH7.2-7.4]. Each single-cell suspension cell concentration was counted using Flow-count fluorospheres (Beckman Coulter, Brea, CA, USA) and a CytoFlex S flow cytometer (Beckman Coulter).  Single-cell suspensions were subjected to a BD Rhapsody system with BD Rhapsody Targeted & Abseq Reagent kit (BD Biosciences) following the manufactures instructions. 20000-30000 cells were subjected to the BD Rhapsody workflow. After reverse transcription, Exonuclease I treatment of the resultant BD Rhapsody beads was performed at 37C for 60 minutes with 1,200 rpm on a Thermomixer C with Thermotop. Resultant beads were immediately chilled on ice; the supernatant was removed and washed with 1 mL of WTA wash buffer [10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 0.02% Tween-20], 200 L of BD Rhapsody lysis buffer, once with 1 ml of WTA wash buffer, twice with 500 L of WTA wash buffer, resuspended with 200 L of Beads resuspension buffer. During the washing step, beads-containing DNA LoBind tubes (Eppendorf Japan) were replaced twice. Exonuclease I-treated BD Rhapsody beads were subjected to TAS- Seq workflow for cDNA library amplification (Shichino S., et al Commune Biol 2022). 150 ng of TAS-Seq amplified cDNA libraries was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina. Ligated products were purified by AmPure XP beads and 9 cycles of Barcoding PCR were performed. Resultant products were purified twice by double size selection with 0.5  0.3 (final 0.8) AmPure XP beads and eluted with 30 L of 10 mM Tris-HCl pH8.0. Size distribution of amplified products was analyzed by MultiNA system with appropriate dilutions. Resultant libraries and barcoded hashtag libraries were quantified using the KAPA Library Quantification Kit. Pooled libraries were sequenced by Illumina Novaseq 6000 sequencer. Pair-end Fastq files (R1: cell barcode reads, R2: RNA reads) of TAS-Seq and BD WTA kit data were processed as follows. Adapter trimming of sequencing data was performed using cutadapt 3.4. Filtered reads were chunked into 16 parts for parallel processing by using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.7. Reference RNA sequences were built by concatenating cDNA and ncRNA fasta files of the Ensembl database (build GRCm38 release-101 for mouse data and GRCh38 release-101 for human data). Associated cDNA reads were mapped to reference RNA using bowtie2-2.4.2 by the following parameters: -p 2 --very-sensitive-local -N 1 -norc -seed 656565 -reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files by the python script and pysam 0.15.4 (https://github.com/pysam-developers/pysam), and read counts of each gene in each cell barcode were counted using mawk. Resultant count data was converted to a single-cell gene-expression matrix file. The inflection point of the knee-plot (total read count versus the rank of the read count) was detected using DropletUtils package48 in R-4.1.2 (https://cran.r-project.org/). Cells of which total read count was over inflection point were considered as valid cells.