GSM5136171: Multiplexed05_Tumor_NoGEMy_GEX; Mus musculus; RNA-Seq
Lungs were perfused with PBS and inflated with digestion medium (HBSS supplemented with 1 mg/mL collagenase I, 20 μg/mL DNAse I and Dispase II). Single-cell suspensions were prepared by finely mincing tissues with a scalpel and incubating the tissue on a shaker at 37°C for 20 minutes in 1 mL of digestion media. Tissue was passed through a 70 μM mesh strainer and washed with collection media twice. Tumors were disassociated using a modified protocol as described (Beury et al. 2016). A fragment of the tumor was placed into a gentleMACs C tube (Miltenyi Biotech) containing 5 mL of digestion media. Tumors were then minced with scissors, processed on the gentleMACS dissociator (Miltenyi Biotech) using the program m_impTumor_02. Tubes were secured in an inverted position and agitated in a 37°C shaker at 100 rpm for 40 minutes. Samples were processed again on the gentleMACS dissociator using program m_impTumor_03, passed through 70 μM cell strainer, and washed with collection media. For spleens and lymph nodes, tissues were mashed through a 70 μM cell strainer and washed with collection media. For spleen and tumor tissues, red blood cells were lysed with ACK lysis buffer (Life Technologies) for 5 minutes and washed with collection media. Cell suspensions were labeled with TotalSeqA hashtag oligo antibodies (BioLegend) before being combined for multiplexed loading on the 10x Genomics Chromium instrument using 3' v3 chemistry Libraries were constructed according to 10x Genomics User Guide (Chromium 3' v3) with a modification to include antibody cell labeling barcode libraries according to Stoeckius et al, 2018