GSM9370660: Lung Cells, KO-72 hour post pneumococcal infection; Mus musculus; RNA-Seq
Lungs were perfused with 10mL heparin-PBS, then removed. Lungs were then perfused with 500ul of digestion buffer ( HBSS, 0.3 mg/mL Liberase and 157 U/ul DNase I), and transferred to a C-tube (Miltenyi Biotech) for mechanical disassociation using gentleMACS (Miltenyi Biotech), default program m_lung_01. Samples were then placed in a shaking incubator for 15 minutes at 37oC. Mechanical disassociation was repeated using the gentleMACS using default program m_lung_2.  Reaction was neutralized with 6.5 mL of ice-cold FA3 buffer (PBS, 10mM HEPES, 2mM EDTA, 1% FBS).  Cells were filtered using a 70 um cell strainer.  Strainer was rinsed with 2mL FA3 buffer. Cells were then pelletted by centrifugation (300xg for 5 minutes at 4oC). Supernant was discarded and RBC lysis was performed by adding 1mL of 1x RBC Lysis buffer (BioLegend). Cells were mixed by pipetting and incubated at room temperature for 1 min.  Reaction was neutralized by adding 7 mL of FA3 buffer. Cell suspension was filtered over a 40 um cell strainer and rinsed with 2 mL FA3 buffer.  Cells were again pelleted and supernant discarded.  Dead cells were removed using Dead Cell Removal kit (Miltenyi Biotech) according to manufacture protocol.  Cells were transferred into FACS buffer (PBS, 2% FBS, 0.1% sodium azide). Viability and counts were assessed using TC-20 cell counter (BioRad) and 1x10^6 cells/mL were submitted for scRNA-seq. Lungs from 1 mouse per treatment group were processed as previously described.Briefly, the lungs were perfused with 10 mL of heparin-PBS. The GentleMACS dissociator (Miltenyi Biotech) was used to separate the harvested lungs from the digesting solution (collagenase I, 0.2 μg/μL + DNase I, 75 U/mL + heparin, 1.5 U/mL) in Dulbecco's Modified Eagle's Media (DMEM). Samples were shaken for 30 minutes at 37oC.  PBS with 4 mM EDTA was used to counteract the activity of the digestion solution. Red blood cells were lysed for 1 minute in 1 mL of lysis solution (BioLegend), and then neutralized with ice-cold Gibco DMEM. Dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec). FACS buffer (2% fetal bovine serum (FBS) + 0.1% NaN3 in PBS) was used to prepare cells for RNAseq. All reagents, unless specified, were purchased from Sigma. The viability and quantification of single-cell suspensions obtained from the whole lung were determined using a LUNA-FLTM Dual Fluorescence Cell Counter (Logos Biosystems). Following manufacturer recommendations, single cells were extracted from cell suspensions (100–2,000 cells/μL) using a 10x Chromium controller (10x Genomics). The gel beads emulsion (GEM)/sample solution was collected and placed into strip tubes. Following cDNA amplification, reverse transcription was carried out using a thermocycler (C1000 TouchTM Thermal Cycler, Bio-Rad). Amplified products were solid phase reversible immobilization (SPRI) bead-purified and evaluated by a Fragment Analyzer (Agilent, Santa Clara, CA). Twenty-five percent of the cDNA volume was fragmented, and PCR purification and clean-up were carried out using double-sided SPRIselect (Beckman Coulter). Following adaptor ligation, each sample underwent SPRI clean-up and PCR amplification utilizing sample-specific indexes. The Fragment Analyzer was used to quantify, purify, and determine the library size distribution of PCR products. PCR products were purified, quantified and sequenced on an Illumina (San Diego, CA, USA) Novaseq6000. Libraries were sequenced to an average depth of 50,000 reads per cell following manufacturer-recommended conditions.