GSM9046254: IMM, GoodLearner, rep3; Gallus gallus; RNA-Seq
Frozen IMM from the left hemisphere was homogenized in a Nuclei PURE Lysis Buffer, (NUC-201, Sigma, formulated for solid tissues) supplemented with DTT, Triton X-100, protease inhibitors and 0.5% RNasin Plus RNase inhibitor (Promega).  The procedure was carried out in a 1 ml Dounce homogenizer (Wheaton) using 10 strokes of the loose Dounce pestle followed by 10 strokes of the tight pestle to liberate nuclei. Homogenate was strained through a 30 μm cell strainer (Miltenyi Biotech) and centrifuged at 900 x g for 10 minutes to pellet nuclei. The supernatant was carefully removed. The pellet was resuspended in homogenisation buffer and strained through a 20 μm cell strainer. The filtered suspension was centrifuged at 900 x g for 10 minutes to pellet nuclei. Further separation of nuclei from myelin debris was carried our as described by Zhou et al, (2020). Nuclei pellets were resuspended in 500 uL Nuclei Wash buffer (1% BSA in PBS with 0.2 U/uL RNasin (Promega) and 900 uL 1.8 M Sucrose. This combined 1400 uL mixture was carefully layered on 500 uL 1.8 M sucrose and centrifuged at 13,000 x g for 45 min at 4 oC. The nuclear pellet obtained was strained through a 30 μm cell strainer, resuspended in Foetal bovine serum + DMSO (90% +10%) and stored for further experiments at -70 0C. Frozen nuclei suspensions were thawed, washed in a small volume of PBS/BSA/RNase inhibitor at 500 g centrifugation and strained again. Libraries were prepared with a Chromium Controller and the Chromium Single Cell 3p RNA library v3 chemistry (10X Genomics, USA), according to the manufacturer's instructions.