GSM5642600: C1q_treated_lung; Mus musculus; RNA-Seq
Murine lungs were isolated from C1q-administrated WT mice. Lungs were minced into 0.5 mm2 with a razor blade and digested with Liberase solution [RPMI-1640 (Nacalai Tesque) supplemented with 10% FBS (MP Bio Japan), 10 mM HEPES pH7.2-7.4 (Cytiva (Global Life Sciences Technologies Japan)), 0.25 mg/ml Liberase TM (Roche), and 2 kU/mL DNase I (Merck)] at 37 ºC for 60 minutes. Cell suspensions were washed and resuspended with 25% Percoll PLUS (Cytiva). After under layered 65% Percoll PLUS, cell suspensions were centrifuged at 20ºC for 1,000 ×g for 20 minutes, and the middle layer was collected. Resultant cell suspensions were washed and resuspended with 1000 μL of preparation medium [RPMI-1640 supplemented with 5% FBS and 10 mM HEPES]. Each single-cell suspension cell concentration was counted using Flow-count fluorospheres (Beckman Coulter) and a Gallios flow cytometer (Beckman Coulter).Cells were stained with 2.5 μg/ml of Totalseq anti-mouse Hashtag-A antibodies (BioLegend) at 4ºC for 30 minutes and washed thrice with Cell Staining Buffer (BioLegend) and pooled. Pooled single-cell suspensions were subjected to a BD Rhapsody system with BD Rhapsody Targeted & Abseq Reagent kit (BD Biosciences) following the manufacture's instructions. 20000 cells were subjected to the BD Rhapsody workflow. After reverse transcription, Exonuclease I treatment of the resultant BD Rhapsody beads was performed at 37ºC 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 0.05% Tween-20], 200 μL of BD Rhapsody lysis buffer (for inactivation of enzyme), once with 1 ml of WTA wash buffer, twice with 500 μL of WTA wash buffer, resuspended with 200 μL 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 and/or hashtag library amplification (Shichino S., bioRxiv, 2021). 150 ng of 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 μL 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.
GEO Accession
GSM5642600