title: GSM8791792: Control, replicate2, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM8791792
library_construction_protocol: For micro-dissected hypothalamic arcuate-median eminence complex (ARC–ME) samples, 4 pairs of postnatal day 0 CTRL and NKO mice were dissected, then brains were rapidly extracted and cooled in ice-cold Hibernate-A medium. To obtain coronal sections containing the ARC–ME region, each brain was immersed in melted 3% low melting point agarose dissolved in DMEM kept at 37°C in a custom cryo mold. The agarose mold was placed on ice for 5-10 mins to solidify. Slicing was performed using a Leica VT 1200S vibratome in ice-cold DMEM:F12 medium at 0.1 mm/s speed and 1 mm vibration amplitude. The 250 μm-thick brain slices were separated from the agarose by gentle pipetting and the continuous six slices from around Bregma -0.96 to – 2.80 mm were collected. The ARC–ME was micro-dissected by knife cuts at its visually approximated dorsolateral borders and pooled by each mouse. Pooled ARC–ME samples were digested by papain solution for 30 min at 37 °C with gentle agitation. After washing with 5 ml Hibernate A medium, the tissues were triturated with fire-polished glass Pasteur pipettes into a single-cell suspension within Hibernate A medium. To remove debris, the single-cell suspension was loaded on a 2-layer gradient and centrifuged at 100 g for 6 min at 4C, and cells were filtered through a 70-μm cell strainer in (Hughes et al., 2020)DPBS with 0.01% BSA (w/v). A 10 μl cell suspension was stained with Trypan Blue and the live cells were counted using an automated cell counter (Invitrogen) and manual. Single-cell libraries were constructed as per the Dropseq protocol (Macosko et al., 2015) with minor modifications. Briefly, each sample was loaded onto 2-3 droplet generation runs, and cell suspensions and droplets were kept on ice prior to droplet encapsulation and droplet breakage. After exonuclease I treatment, samples were performed with second strand synthesis as previously described(Hughes et al., 2020). In the cDNA PCR amplification stage, around 4,000 beads were combined in a single PCR tube reaction and purified individually using 0.6x AMPure XP beads. cDNA across multiple runs of the same sample were pooled together and used as input for tagmentation, where the tagment reaction time was increased to 6 min. Final samples were purified twice with 0.6x AMPure XP beads. Sequencing library fragment sizes were quantified by bioanalyzer, and concentrations were quantified by qPCR (KAPA). Samples were pooled, loaded at 2.0-2.2 pM, and sequenced on a Next-Seq 550 (Illumina) with a 20 bp Read 1 and 64 bp Read 2. The custom Read 1 primer was spiked into the usual Illumina sequencing primer well at the manufacturer's recommended concentration.