GSM8524882: Batch 2; sorted Rabies + nuclei from ARC/DMH/PVH; condition FASTED; Mus musculus; RNA-Seq
Once all samples for each batch were collected, tissue samples were thawed on ice and pooled by region and feeding condition. Tissue was dounce homogenized and purified by density gradient centrifugation into a single-nuclei suspension as previously described 26,94, but with the following modifications: in batch 2, TruSeq anti-Nuclear Pore Complex Proteins Hashtags were applied to track the brain region of origin for each cell nuclei (BioLegend, catalog # 682205, 682207, 682209). We counterstained nuclei with a far-red DNA fluorescent intercalator, DRAQ5 (Thermo Fisher, catalog # 62251, 1:500 dilution in sample) and kept them chilled on ice until sorting. We sorted single, DRAQ5+/mCherry+ nuclei using the Becton Dickenson FACS Aria Fusion (sample batch 1) or Becton Dickenson Influx (sample batch 2) cell sorters. All sorts were performed with SCYM (ASCP) certified technical assistance at the University of Virginia Flow Cytometry Core, using an 85 μm nozzle and set to purity mode. To separate nuclei from non-nucleated debris, we gated for events with high relative intensity of DRAQ5 fluorescence, using a 640 nm excitation and a 670/30 nm collection filter. We then gated the nuclei by forward scatter area vs. side scatter area to eliminate large aggregates, followed by forward scatter area vs. forward scatter height to select for singlets. Finally, from the single nuclei, we selected mCherry+ nuclei based on their high relative fluorescence, excitation 561 nm and 610/20 nm collection filter. We sorted mCherry+ nuclei into 18.8 μL of RT Reagent B from the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1 We added the remainder of the kit Step 1 master mix reagents to the capture tube, plus enough resuspension buffer to reach a total volume of 75 μL. We then processed the sample into complementary DNA (cDNA) sequencing libraries according to the manufacturer's instructions (10X Genomics, CG000204 Rev D). Hashtag oligonucleotide (HTO) libraries were prepared according to a previously published protocol, with the following modification: double-sided SPRI (solid phase reversible immobilization) was repeated for a total of two times to cleanly separate HTO libraries from cDNA libraries after amplification and, if needed, just prior to pooling libraries for sequencing. Libraries were sequenced at a concentration of 1.85 pM and with a 75-cycle, high-output kit on the Illumina NextSeq 550 according to the manufacturer's instructions..