GSM2887141: Single P7 OB mouse dopaminergic neuron (TH_016_E12); Mus musculus; RNA-Seq
Resected brain tissues were dissociated using papain (Papain Dissociation System, Worthington Biochemical Corporation; Cat#: LK003150) following the trehalose-enhanced protocol reported by Saxena, et. al, 201217 with the following modifications: The dissociation was carried out at 37oC in a sterile tissue culture cabinet. During dissociation, all tissues at all timepoints were triturated every 10 minutes using a sterile Pasteur pipette. For E15.5 tissues, this was continued for no more than 40 minutes. For P7, this was continued for up to 1.5 hours or until the tissue appeared to be completely dissociated. Additionally, for P7 tissues, after dissociation but before cell sorting, the cell pellets were passed through a discontinuous density gradient in order to remove cell debris that could impede cell sorting. This gradient was adapted from the Worthington Papain Dissociation System kit. Briefly, after completion of dissociation according to the Saxena protocol, the final cell pellet was resuspended in DNase dilute albumin-inhibitor solution, layered on top of 5 mL of albumin-inhibitor solution, and centrifuged at 70g for 6 minutes. The supernatant was then removed.  For each timepoint-region condition, pellets were resuspended in 200 μL of media without serum comprised of DMEM/F12 without phenol red, 5% trehalose (w/v), 25 μM AP-V, 100 μM kynurenic acid, and 10 μL of 40 U/μl RNase inhibitor (RNasin® Plus RNase Inhibitor, Promega) at room temperature. The resuspended cells were then passed through a 40 uM filter and introduced into a FACS machine (Beckman Coulter MoFlo Cell Sorter or Becton Dickinson FACSJazz). Viable cells were identified via propidium iodide staining, and individual neurons were sorted based on their fluorescence (EGFP+ intensity, See Figure S4B) directly into lysis buffer in individual wells of 96-well plates for single-cell sequencing (2 μL Smart-Seq2 lysis buffer + RNAase inhibitor, 1 μL oligo-dT primer, and 1 μL dNTPs) according to Picelli et al., 201418. Blank wells were used as negative controls for each plate collected. Upon completion of a sort, the plates were briefly spun in a tabletop microcentrifuge and snap-frozen on dry ice. Single cell lysates were subsequently kept at -80°C until cDNA conversion. Library preparation and amplification of single-cell samples were performed using a modified version of the Smart-Seq2 protocol18. Briefly, 96-well plates of single cell lysates were thawed to 4°C, heated to 72°C for 3 minutes, then immediately placed on ice. Template switching first-strand cDNA synthesis was performed as described above using a 5'-biotinylated TSO oligo. cDNAs were amplified using 20 cycles of KAPA HiFi PCR and 5'-biotinylated ISPCR primer. Amplified cDNA was cleaned with a 1:1 ratio of Ampure XP beads and approximately 200 pg was used for a one-quarter standard sized Nextera XT tagmentation reaction. Tagmented fragments were amplified for 14 cycles and dual indexes were added to each well to uniquely label each library. Concentrations were assessed with Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and samples were diluted to ~2 nM and pooled. Pooled libraries were sequenced on the Illumina HiSeq 2500 platform to a target mean depth of ~8.0 x 105 50bp paired-end fragments per cell at the Hopkins Genetics Research Core Facility.
GEO Accession
GSM2887141