Rhapsody scrna seq platform

Surgically resected NSCLC tumor tissues and adjacent normal tissues were minced into small pieces (<1 mm) on ice and enzymatically digested with agitation for 30 min at 37°C using the BD TuDoR™ dissociation reagent (BD Biosciences). The obtained single-cell solution was sieved through a 70 μM cell strainer (Corning) and red blood cells were removed using the BD Pharm Lyse™ lysing solution (BD Biosciences). Cells were counted and viability assessed with the BD Rhapsody scRNA-seq platform (BD Biosciences) using Calcein-AM (Invitrogen) and Draq7 (BD Biosciences).

Surgically resected NSCLC tumor tissues and corresponding benign lung tissues were minced into small pieces (<1 mm) on ice and enzymatically digested with agitation for 30 min at 37 °C using the BD TuDoR™ dissociation reagent (BD Biosciences). The obtained single-cell solution was sieved through a 70 μM cell strainer (Corning) and red blood cells were removed using the BD Pharm Lyse™ lysing solution (BD Biosciences). Cells were counted and viability assessed with the BD Rhapsody scRNA-seq platform (BD Biosciences) using Calcein-AM (Invitrogen) and Draq7 (BD Biosciences). Immediately, >1 × 10⁶ cells of the obtained single-cell suspensions were subjected to the sample-tag staining procedure (20 min RT, 3x washing by 5 min centrifugation at 400 rpm). Total RNA was isolated before (T1) and after (T2) the sample-tag staining procedure using the RNeasy Mini kit (Qiagen) and RNA quality (RNA integrity number, RIN) was assessed with the High Sensitivity RNA ScreenTape assay (Agilent) and the 4200 TapeStation (Agilent) system according to the manufacturer's instructions.

Liver biopsies taken pre (T0) and at the end (T1) of NMP as well as after reperfusion (T2) (n = 8 livers) were immediately minced into small pieces (<1 mm) on ice and subsequently digested enzymatically for 10 min at 37 °C with agitation using the BD TuDoR dissociation reagent (BD Biosciences). The single-cell suspension was filtered through a 100 µM cell strainer and red blood cells were removed with the BD Pharm Lyse (BD Biosciences) lysing solution according to the manufacturer´s protocol. Cell viability was measured with the BD Rhapsody scRNASeq platform (BD Biosciences) using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences).