title: GSM5852254: Bone marrow-derived monocytes from healthy mice replicate 3 [RNA-seq]; Mus musculus; RNA-Seq
library_construction_protocol: Normal mammary fat pad: Tissue was minced using scissors and placed and disrupted for 25 min at 37oC in a gentleMACS C tube using gentleMACS dissociator, in the presence of enzymatic digestion solution containing 1 mg ml-1 collagenase II, 1 mg ml-1collagenase IV and 70 unit ml-1 DNase. Primary tumor: Tissue was minced using scissors and treated with enzymatic digestion solution containing 3 mg ml-1 collagenase A and 70 unit ml-1 DNase in RPMI 1640 for 20 min at 37oC, with pipetting every 3 min. Foe BMDM extraction the Tibia and Femur leg bones were dissected Bones were flushed with MACS buffer using a 5 ml syringe with a 25 G needle. The flushed cells from the bone marrow were centrifuged at 500 g for 5 min and resuspended in red blood cell lysis buffer  cells were washed with PBS and resuspended in macs buffer. 3' end mRNA libraries were prepared for sequencing using a method developed at Dr. Ido Amit's lab, Weizmann Institute of Science 3' RNA-seq for digital gene expression quantitation; Single end protocol R2 used for 7N barcode + 8N UMI concatenated to R1 header