GSM5255536: DMV neuron nucleus 366; Mus musculus; RNA-Seq
The following morning, the RNAlater-preserved tissue samples were processed into single-nuclei suspensions according to the sNuc-Seq as previously described (Todd et al., 2020). Briefly, the pooled DMV tissue samples were dounce homogenized (Wheaton tissue grinders, catalog # 357538) and separated by density gradient centrifugation. Each pellet was then re-suspended in 0.5 ml sNuc-Seq resuspension buffer and filtered through a 20 μm mesh (Miltenyi Biotec pre-separation filters, catalog # 130-101-812) to remove clumped nuclei and larger debris. Nuclei were counterstained with two drops of NucBlue Live ReadyProbes Reagent (ThermoFisher Scientific catalog # R37605) per ml of nuclei. Nuclei were sorted by MoFlo Astrios EQ cell sorter (Harvard Bauer core facility), gating for NucBlue+/mCherry+ singlets and distributing them individually to wells of 96-well plates containing 5 μl per well of nuclei capture buffer (1% beta-mercaptoethanol in Qiagen Buffer TCL; catalog # 1031576). Each plate was loaded with single nuclei, one per well. Loaded plates were centrifuged for 5 min at 2,500 xg and 4degC to ensure nuclei reached the capture buffer and frozen at -80degC for up to 8 weeks until use. Sample plates were transferred to an RNA-dedicated PCR cabinet, thawed quickly, and immediately purified with RNA-Clean XP (Beckman Coulter, catalog # A63987; 2.5:1 ratio of RNA-Clean XP to sample volume), eluting in 4μl Smart-seq2 buffer (2 μl Smart-seq2 cell lysis buffer, 1 μl 100 μM oligo-dT primer, and 1 μl 10 mM dNTP mix). cDNA was then generated and amplified as described in the Smart-Seq2 protocol, except that 22 PCR cycles were used for the amplification step. After the PCR step, 10 μl of each sample was transferred to new 96-well plates for subsequent processing. Amplified cDNA was purified with two rounds of SPRIselect (Beckman Coulter, catalog # B23318; 0.8:1 ratio of SPRIselect to sample volume), eluting in ultrapure water. cDNA concentration was measured by Qubit fluorometry in a microplate format, each well containing 2 μl of cDNA sample and 98 μl of Qubit working solution (ThermoFisher Scientific, catalog # Q32854). Qubit readings were collected with a fluorescence microplate reader (Molecular Devices SpectraMax M5; Harvard Medical School ICCB-Longwood core facility) and used to calculate DNA concentrations by comparing each sample's Qubit reading to standards in a 50% dilution series (n=8 standards; range, 0.25 ng/μl to 0.01 ng/μl plus a negative control, 0.0 ng/μl). cDNA samples were then diluted to 0.15 ng/ μl with ultrapure water and processed into sequencing libraries with sample-specific Nextera indices and the Nextera DNA Sample Prep Kit and Nextera XT Index Kit v2 sets A-D (Illumina, catalog #s FC-131-1096, FC-131- 2001, FC-131-2002, FC-131-2003, FC-131-2004, respectively). Nextera XT was used at 1/4 reaction volumes but otherwise according to manufacturer's instructions. Sequencing libraries were purified with two rounds of SPRIselect (0.8:1 ratio of SPRIselect to sample volume) and eluted in a single volume of ultrapure water. Library pool concentration was measured by Qubit according to the manufacturer's instructions, sized using Agilent BioAnalyzer high sensitivity kit, and then diluted with ultrapure water to a size-corrected concentration of 2 nM. The diluted library pool was denatured and further diluted to 2 pM loading concentration according to Illumina NextSeq recommendations, then sequenced with a NextSeq 500 v2 75cycle sequencing kit (Illumina, catalog # FC-404-2005). Smart-Seq2