GSM7316425: Lacrimal Gland, Male, replicate 2; Mus musculus; RNA-Seq
One female and two male samples were prepared for scRNAseq analysis. For each sample, six dissected LGs of three animals aged of 2 months were combined and processed as follows. First, LGs were quickly minced on ice in a dish pre-filled with cold DPBS, rinsed, and transferred to a 12-well plate placed in a shaking water bath (37°C, 80 rpm) containing 2 mL of digestion medium of following composition: DMEM/F12 , 0.5% BSA , 15 mM HEPES, 3 mM CaCl2 , 125 U/mL collagenase IV , 1 U/mL dispase II  and 20 U/mL DNase-I. After 30 min, samples were gently triturated by pipetting up and down 15 times with a wide-bored 1 mL tip. At 60 and 90 min, samples were gently triturated 10 to 15 times with a regular 1 mL pipet tip. At the end of the digestion (90 min in total), cells were pelleted (300 x g, 5 min, 4 °C), washed with cold HBSS buffer supplemented with 10 mM HEPES and 1 mM EDTA and gently re-suspended in 1 mL of accutase solution. After 5 min at 37°C, cell suspension was pipetted up and down 5 times and left at RT for 2 min. Then, accutase was inactivated by the addition of 1 mL of DMEM/F12 supplemented with 10% FBS and 15 mM HEPES. Cells were pelleted (300 x g, 5 min, 4 °C) and incubated for 10 min at RT with HBSS supplemented with 10 mM HEPES, 2 mM MgCl2, 2 mM CaCl2 and 200 U/mL DNase-I. Cell suspension was sequentially passed 2 times through a 3 mL syringe with needles: 18G, 22G and 25G. Dissociated cells were filtered through a pre-wet 70-µm mesh cell strainer and pelleted (300 x g, 5 min, 4 °C) before incubation for 5 min at RT with 5 mL of RBC lysis buffer 1X  to deplete erythrocytes. After centrifugation (300 x g, 5 min, 4 °C) and re-suspension into cold HBSS supplemented with 0.5% BSA and 10 mM HEPES, cells were passed through a pre-wet 30-µm mesh cell strainer. After centrifugation (400 x g, 5 min, 4 °C), dead cells and debris were depleted using the MS columns and the dead cell removal kit . The cell suspension was washed 3 times with cold scRNA-seq buffer composed of DPBS supplemented with 0.04% BSA, and pelleted at 400 x g, 5 min, 4 °C. The number of cells and the viability rate (78-79%) were evaluated using trypan blue staining analyzed with the Countess 3. Cell suspension volumes were then adjusted with cold scRNA-seq buffer to obtain between 700 and 1200 cells per µL and cells were counted again to obtain the final cell density. Cells were kept on ice at all times. Cells were processed for scRNA-seq using the Chromium Single Cell 3' kit (v3.1 Chemistry) from 10X Genomics (Pleasanton, CA) according to the manufacturer's protocols. cDNA was amplified by 11 cycles. Analysis of cDNA and libraries was done using Bioanalyzer or TapeStation (Agilent).