GSM6859169: adult male arcuate nucleus of the hypothalamus, sham, rep 1; Mus musculus; RNA-Seq
ARC brain punches were homogenized using a pestle mixer (Argos Technologies) in Nuclei EZ lysis buffer (Sigma-Aldrich). Homogenates were then filtered through a 70 μm cell strainer (Sigma-Aldrich) and collected in a fresh Eppendorf tube, followed by two rounds of centrifugation at 500 g for 5 mins at 4°C in Nuclei EZ lysis buffer. After removal of supernatant, 500 μL of Nuclei Wash and Resuspension buffer (NWR, 1.0% bovine serum albumin, 0.2 U/μl RNase inhibitor in phosphate buffered saline) was added to the pellets and incubated on ice for 5 mins without pellet disruption. Pellets were then resuspended in an additional volume of 500 μL NWR. An additional three rounds of centrifugation were carried out in the presence of NWR. After the last centrifugation step, the pellets were resuspended in 50 μL of NWR and the number of isolated intact nuclei was counted using a hemocytometer. Sequencing libraries were constructed following the manufacter's instructions using the Chromium Signle Cell3' GEM, Library & Gel Bead Kit v3 (10X Genomics; https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf). Briefly, GEMs are generated by combining barcoded Single Cell  3ʹ v3.1 Gel Beads, a Master Mix containing cells, and Partitioning Oil onto Chromium Next GEM Chip G. Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any copartitioned cell is lysed. Primers are mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA. GEMs are then broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. TruSeq Read  1 (read 1 primer sequence) is added to the molecules during GEM incubation. P5, P7, a sample index, and TruSeq Read  2 (read  2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.