GSM4083924: Brainstem Recovery Sleep; Mus musculus; RNA-Seq
The single cell suspensions preparation procedure for all the three brain regions was adapted from Holt and colleagues. Briefly, tissues were dissociated with Papain in the manufacturer supplied medium followed by manual trituration using a fire polished silanized Pasteur pipette and filtration through 70µm cell strainer. Cells were pelleted at 300 x g, 10 min, the supernatant was discarded, and cells resuspended in a minimal volume of PBS with 0.5% of BSA. To reduce the debris, we incubated the cell suspensions with myelin removal beads and passed it through an LS Column with 70µm pre-separation filter. Flow-through containing single cell suspension were collected and estimated the yield and viability. Single cell suspension were diluted in PBS with 0.5% of BSA to obtain ~ 350 cells/ µl and followed the 10X Genomics Chromium Single Cell Kit Version 2 manual for the capture, cDNA synthesis, library preparation, and sequencing.