GSM4783934: Splenocyte_10x_scRNA-seq; Mus musculus; OTHER
We dissected mice and harvested the spleens on ice in (1x PBS Gibco, 10010-023). We washed spleens in RPMI media (Gibco, 22400-089) and using a scalpel individually cut the spleens into small pieces. We digested the tissue with a 10x collagenase D (Sigma Aldrich 11088866001) / DNase I (Qiagen, 79524) solution (0.05g collagenase D and 10ul DNase I in 1ml of 1 x PBS) diluted to 1x in RPMI by incubation at 37°C shaking (400 rpm) for 30 minutes. We transferred the dissociated tissue onto a 70µm cell strainer (Falcon, 352350) and carefully used a 3ml syringe plunger (BD, 309578) to compress the tissue through the filter. The filtered solution was centrifuged for 10 minutes at 300g and 4°C and we resuspended and incubated the subsequent pellet in ACK lysis buffer (Gibco, A1049201) for 10 minutes at room temperature to lyse the red blood. We quenched the reaction with RPMI media and centrifuged the cells again. We washed twice with cold PBS and used this for final resuspension. We filtered the cells through a 40µm filter (pluriSelect, 43-50040-01) prior to counting the cells using a disposable hemocytometer (INCYTO, DHC-N01-2). We controlled for cell viability using a live/dead stain (Invitrogen, S34860). 10x Chromium Single Cell 3' v3