title: GSM8415922: Batch 6; sample 20; SpinalTRAP-negative VLM cells; Mus musculus; RNA-Seq
library_construction_protocol: We then processed brain tissue into cell nuclei suspensions using one of two protocols, as described below. Importantly, tissue for all sample batches was dissected using the same approach, though the postdissection processing of tissue samples differed between batches 1-2 and batches 3-6, as described below (details provided in table 1). For sample batches 1 and 2, we transferred the dissected tissue to an RNase-free microcentrifuge tube, froze it in a slurry of dry ice and methanol, and then stored at -80 ℃ for less than a month. After thawing a batch of tissue samples on ice, we isolated cell nuclei from the thawed tissue using a previously described hypotonic lysis protocol (Matson et al., 2018), modified as follows: trituration with 5 Pasteur pipettes, starting with an unpolished pipette, followed by a series of four pipettes with tips fire polished to progressively smaller diameters; 5 to 10 strokes for each size pipette, or until the tissue passed smoothly through the pipette; and filtered resuspended nuclei with 20 µm strainer (pre-separation filter; catalog # 130-101-812; Miltenyi Biotec; Bergisch Gladbach, Germany). We fluorescently labeled nuclei with propidium iodide and then quantified them with a DeNovix CellDrop automated cell counter. a 20 µm cell strainer (Miltenyi Biotec pre-separation filter), fluorescently labeled DNA with NucBlue Live ReadyProbes Reagent (catalog # R37605, Thermo Fisher Scientific), and sorted the nuclei on a Becton Dickenson Influx cell sorter equipped with an 85 µm nozzle and set to purity mode. All sorting was performed with a Specialist in Cytometry (SCYM; ASCP) certified technical assistance at the University of Virginia Flow Cytometry Core. To separate nuclei from non-nucleated debris, we gated for events with high relative intensity of NucBlue fluorescence. 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 H2B-mCherry+ and -negative nuclei based on their high and low relative fluorescence, respectively, with 561 nm excitation and 610/20 nm collection filter. We separated H2BmCherry+ and -negative nuclei singlets into low-bind microcentrifuge tubes, each containing 18.8 uL of RT Reagent B (from Chromium Next GEM Single Cell 3ʹ v3.1 kit). For sample batches 3 through 6, which involved fluorescence-activated cell sorting (FACS), we isolated cell nuclei from preserved RVLM tissue samples based on a published protocol (Habib et al., 2016; Todd et al., 2020). RVLM tissue was microdissected as described above, but then transferred to RNase-free microcentrifuge tubes containing ice-cold RNAprotect reagent (catalog # 76104; Qiagen; Venlo, The Netherlands). We stored the tissue samples in RNAprotect at 4℃ overnight, then removed the liquid and transferred the preserved tissue samples to -80 ℃ storage for no more than 3 months. When ready to proceed, we thawed a batch of tissue samples on ice, homogenized them in 1 mL of homogenization buffer 25 times with pestle A and 25 times with pestle B (Kimble Kontes Dounce Tissue Grinder, DWK Life Sciences, catalog #885300-0001), then separated the nuclei from debris by density gradient centrifugation according to the protocol (Habib et al., 2016). We filtered the resuspended nuclei through NucBlue Live ReadyProbes Reagent (catalog # R37605, Thermo Fisher Scientific), and sorted the nuclei on a Becton Dickenson Influx cell sorter equipped with an 85 µm nozzle and set to purity mode. All sorting was performed with a Specialist in Cytometry (SCYM; ASCP) certified technical assistance at the University of Virginia Flow Cytometry Core. To separate nuclei from non-nucleated debris, we gated for events with high relative intensity of NucBlue fluorescence. 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 H2B-mCherry+ and -negative nuclei based on their high and low relative fluorescence, respectively, with 561 nm excitation and 610/20 nm collection filter. We separated H2BmCherry+ and -negative nuclei singlets into low-bind microcentrifuge tubes, each containing 18.8 uL of RT Reagent B (from Chromium Next GEM Single Cell 3ʹ v3.1 kit We added the remainder of the Step 1 mastermix components and then proceeded with the Chromium Next GEM Single Cell 3ʹ v3.1 kit (catalog # PN-1000128 or PN-1000121; 10X Genomics; Pleasanton, CA; U.S.A.) according to the manufacturer's instructions (CG000204 Rev D; 10X Genomics), tagging each sequencing library with a different 10X Sample Specific Index (catalog # PN-1000213; 10X Genomics) for sequencing libraries in multiplex.