title: GSM6435660: ANKL1_Liver; Homo sapiens; RNA-Seq
library_construction_protocol: Fresh liver and spleen were harvested from ANKL1-PDX mouse. Both organs were homogenized and filtrated with Falcon 70 µm of cell strainer (Corning). Liver homogenized sample were then treated with 1 mg/mL of collagenase A (Roche) and 100 U of DNAse I (Roche) in 5 mL of MEM-alpha medium (Thermo Fisher Scientific) at 37 °C for 15 minutes, following centrifugation (500xg, 5 minutes) and removal supernatant. liver pellet were then re-suspended with 35% Percoll (Sigma-Aldrich), and centrifuged for 1000xg, 10 minutes following removal supernatant. Liver pellet and spleen homogenized samples were then treated with RBC Lysin Buffer (BD pharm) to eliminate erythrocytes. After centrifugation for 300xg, 3 minutes and removal supernatant, cells were staind with microbeads-conjugated anti-human CD45 (Miltenyi Biotec) and apply for autoMACS system (Miltenyi Biotec). A suspension of cells under designated experimental conditions was added to a polydimethylsiloxane microwell slide containing poly(dT) barcoded beads (BD Bioscience) and allowed to settle into the well via gravity. The cells were lysed, and poly(dT)-barcoded beads with bound cellular mRNA were collected, followed by the synthesis of cDNA. The obtained cDNA was then amplified with an adaptor sequence attached to captured mRNA via the terminal deoxynucleotidyl transferase method. After sonication, the cDNA fragments were subjected to end repair and dA-tailing using NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs) and mixed with an adaptor primer with dT (multiplexing read 2 sequencing primer Illumina TruSeq™; Illumina) and Ligation High Ver.2 (TOYOBO). Libraries were then sequenced using the NovaSeq6000 Sequencing System (Illumina).