alias: GSM4546897
title: GSM4546897: P30_M SMG scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: SMG were dissected with sharp tweezers from 4-7 littermates of E12-E16 ICR mice, and 2-4 littermates of P1 (ICR), P30 (ICR) and adult (C3H mice). E12 and E14 glands were incubated at 37oC for 30 minutes with 500ul of papain (20U/ml in 1mM L-cysteine with 0.5mM EDTA. Glands were dissociated with a pipette in 5-minute intervals. Papain was inactivated with (1:1) albumin-inhibitor solution and the cell suspension was collected by centrifugation at 400g at 4oC for 10 minutes, washed twice and resuspended in Hanks balanced salt solution (HBSS) with 1% BSA.     SMGs from E16 and postnatal day 1 (P1) were isolated and enzymatically dissociated in 5ml of digestion cocktail containing Collagenase II (100mg/ml), Hyaluronidase (50mg/ml), deoxyribonuclease (40mg/ml), and neutral protease (50mg/ml) in HBSS-BSA. Dissociation was performed in a 15ml gentleMACS C tube using a miltenyi gentleMACS Octo Dissociator following their preset dissociation program A (24 seconds of clockwise-counterclockwise spin cycles) followed by 30 min incubation at 37oC. Dissociated cells were centrifuged (400xg) for 10 mins and resuspended in HBSSA-BSA. Then, cells were passed through 100 µm, 40 µm, and 20 µm filters centrifuging (400xg) for 5mins and resuspending in HBSS-BSA after each filtration step. The final cell suspension was centrifuged (400xg) 5 mins and treated with Papain as described above.     For P30 ICR and adult C3H mice, glands were dissociated in a 15ml gentleMACS C tube with 5ml of digestion cocktail prepared with the human tumor dissociation kit (#130-095-929, Miltenyi Biotech, Auburn CA) in RPMI 1640 w/L-Glutamine. 2 SMGs from male and female ICR mice, and 2 SMG from adult female C3H mice were processed individually. Digestion was performed in a Miltenyi gentleMACS Octo Dissociator following the preset 37C_h_TDK_2 program. We added 5ml of RPMI media to the dissociated cells and centrifuged (400xg) for 10 mins. Cells were resuspended in RPMI 1640 w/L-Glutamine with 5% PenStrep (Gibco, USA) and washed twice with RPMI. Next, cells were centrifuged (400g) for 5 minutes and washed with RPMI medium before each filtration step through 70 µm and 40 µm filters. Apart from the enzymatic digestion, all steps were performed on ice. Library preparation was performed according to the manufacturer's instructions for the 10× Chromium single-cell kit v2 and v3 chemistries (10x Genomics). The libraries were then pooled and sequenced on four lanes on a NextSeq500 sequencer (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM4546897