title: GSM5145684: D3-TAN; Mus musculus; RNA-Seq
library_construction_protocol: All murine tissues in this study were prepared from mice sacrificed by cervical dislocation at the same time. For the isolation of bone marrow cells, leg bones from murine femur were flushed using a 21-gauge needle in RPMI1640 medium containing 1% FBS. For the isolation of spleen cells, the whole spleen was dissected and homogenized into single-cell suspensions using a 1 ml syringe plunger with a 21-gauge needle. For the isolation of peripheral blood immune cells, 600~800 ml blood was collected by retro-orbital bleeding. For the isolation of gastric cancer infiltrated immune cells from xenograft tumor tissues (300~1,000mm3) and gastric cancer patient, tumor tissues were dissected into 1~2 mm pieces with scissors in PBS buffer and digested in RPMI 1640 Medium with 1 mg/ml Collagenase II and 100 mg/ml DNase I at 37 °C for 30 min. Subsequently, BM, SP, PB and GC cells were filtered with a 70 mm cell strainer. After a 5 min centrifugation at 500 g, cells were resuspended with red blood cell lysis buffer to deplete red blood cells. Subsequently, cells were washed 2~3 times and stained with fluorophore-conjugated anti-mouse antibodies against Live/dead, CD45, CD11b and Ly6G for 30 min at 4°C. After exclusion of cell doublets and dead cells with, single cells were purified by fluorescence-activated cell sorting (FACS) using a FACS AriaIII cell sorter and Diva software (both BD Biosciences) to achieve > 98% purity. Libraries were constructed using the Single Cell 3' Library Kit V2 (10× Genomics).