GSM9643960: Tumor draining lymph node_ATB_EC; Mus musculus; RNA-Seq
At D11, tdLN (tumor draining lymph node) and tumor were harvested. Excised tumors were cut into small pieces and digested in RPMI medium containing Liberase at 25 mg/ml (Roche) and DNase1 at 150 IU/ml (Roche) for 30 min at 37 °C, and then crushed and filtered twice using 70µm and 30µm cell strainers (Miltenyi Biotec). Lymph nodes were crushed in RPMI medium and subsequently filtered through a 70-µm cell strainer. Tumor cells were enriched using mouse CD45 microbeads from Miltenyi (130-052-30) following the Milteny's protocol. Experimental conditions were pooled prior to loading using the BD™ Mouse Single Cell Sample Multiplexing Kit (Cat No: 633793), with 9 and 11 sample barcodes per cartridge, respectively. Libraries were processed according to the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit (Cat No: 633801) and the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Reagent Kit (Cat No: 665915). Libraries were subjected to indexed paired-end sequencing (2×100 bp) on a DNBSEQ-T7 platform (BGI Genomics, Shenzhen, China) with 30% PhiX spike-in control. A total of 1,415 million reads and 2,068 million reads were sequenced for the tumor and lymph node libraries, respectively.