title: GSM5395695: Experiment 2 - Lvpl3; Mus musculus; RNA-Seq
library_construction_protocol: All tissue fragments were combined, then were treated with Collagenase 1(Sigma Aldrich, Cat. No. LS004196) and 3 (Worthington Biochem, Cat. No. LS004182) at 37˚C for a period of 20 minutes.  Both enzymes were applied at a concentration of 4.2 mg/mL in TESCA buffer (4.75 mL H20: 250 uL 1M TES: 7.2 uL 0.25 M CaCl2).  Then, 100 uL of DNase1 (0.5U/uL) was added and allowed 7 more minutes of incubation at 37˚C.  1.5 mL of DMEM/10% FBS was added to quench enzyme action after this.  The cell suspension was then transferred to room temperature and was triturated slowly and continuously with a 1mL capacity pipette with a wide-bored tip for 5 minutes.  Subsequently, 1 minute was allowed for large pieces of tissue to settle to the bottom of the tube, and supernatant containing the cell suspension was drawn off.  The suspension was centrifuged on a swinging bucket rotor centrifuge for 10 minutes at 100xg.  The cell pellet was resuspended in 220uL of PBS with 0.04% BSA and pressed through a 40um Flowmi cell strainer, leaving 200um of suspended single cell solution. RNA libraries were prepared for sequencing using standard Illumina NovaSeq6000 protocols scRNA-seq (GEM)