| Extract Protocol: |
Mice were euthanized by CO2 inhalation and lungs were immediately perfused with 20 mL ice-cold PBS. Lungs were cut in small pieces with scissors, enzymatically digested at 37°C for 45 minutes with a solution containing collagenase IV (1mg/ml), hyaluronidase (0.1mg/ml) and DNase (4.5mg/ml) (Sigma-Aldrich). Cells were then collected, filtered and red blood cells were lysed at RT for 5 minutes with ACK Lysing Buffer (Lonza). Cells were then collected, filtered and pooled as follows: 3 untreated-chimera, 3 silibinin-treated, 3 baricitinib-treated and 3 vFLIP-tg mice. The cells were washed with PBS 1x, re-suspended in RPMI 1640 medium supplemented with 5% bovine serum albumin and counted. Cell viability was determined by Trypan blue exclusion. For each sample, cells were resuspended in RPMI supplemented with 5% FBS to a final concentration of 106 /ml for single cell analysis.; Mice were euthanized by CO2 inhalation and lungs were immediately perfused with 20 mL ice-cold PBS. Lungs were cut in small pieces with scissors, enzymatically digested at 37°C for 45 minutes with a solution containing collagenase IV (1mg/ml), hyaluronidase (0.1mg/ml) and DNase (4.5mg/ml) (Sigma-Aldrich). Cells were then collected, filtered and red blood cells were lysed at RT for 5 minutes with ACK Lysing Buffer (Lonza). Cells were then collected, filtered and pooled as follows: 3 WT mice, 3 untreated-chimera, 3 silibinin-treated, 3 baricitinib-treated and 3 vFLIP-tg mice. The cells were washed with PBS 1x, re-suspended in RPMI 1640 medium supplemented with 5% bovine serum albumin and counted. Cell viability was determined by Trypan blue exclusion. For each sample, cells were resuspended in RPMI supplemented with 5% FBS to a final concentration of 106 /ml for single cell analysis. |
| Library Construction Protocol: |
Cell suspension were processed using the 10x Genomics Chromium Controller and the Chromium NextGEM Single Cell 3′ GEM, Library& Gel Bead kit v3.1 (Pleasanton, California, United States) following the stand-ard manufacturer’s instructions. In brief, 10,000 live cells were loaded onto the Chromium controller to recover 4,000 single cell GEMs per inlet uniquely barcoded. After synthesis of cDNA, sequencing libraries were generated. Final 10X library quality was assessed using the Fragment Analyzer High Sensitivity NGS kit (Agilent Technologies, Santa Clara, CA, USA) and then sequenced on the Illu-mina NextSeq500 (Illumina, San Diego CA, USA). After synthesis of cDNA, sequencing libraries were generated. Final 10X library quality was assessed using the Fragment Analyzer High Sensitivity NGS kit (Agilent Technologies, Santa Clara, CA, USA) and then sequenced on the Illu-mina NextSeq500 (Illumina, San Diego CA, USA).; Cell suspension were processed using the 10x Genomics Chromium Controller and the Chromium NextGEM Single Cell 3′ GEM, Library& Gel Bead kit v3.1 (Pleasanton, California, United States) following the standard manufacturer’s instructions. In brief, 10,000 live cells were loaded into the Chromium controller to recover 4,000 single cell GEMs per inlet uniquely barcoded. After synthesis of cDNA, sequencing libraries were generated. Final 10X library quality was assessed using the Fragment Analyzer High Sensitivity NGS kit (Agilent Technologies, Santa Clara, CA, USA) and then sequenced on the Illumina NextSeq500 (Illumina, San Diego CA, USA). |
