title: GSM7913148: O, snRNA-seq; Rattus norvegicus; RNA-Seq
library_construction_protocol: Intact hypothalami were cryopreserved in liquid nitrogen from sacrificed rats and approximately 2-3 pooled intact hypothalami in each group were homogenized in 500µL ice-cold homogenization buffer (0.25 M sucrose, 5 mM CaCl2 , 3 mM MgAc2, 10 mM Tris-HCl pH 8.0, 1 mM DTT, 0.1 mM EDTA, 1x protease inhibitor, and 1 U µL−1 RiboLock RNase inhibitor) with Dounce homogenizer. Then the homogenizer was washed with 700µL ice-cold nuclei washing buffer (0.04% bovine serum albumin, 0.2 U/µL RiboLock RNase Inhibitor, 500 mM mannitol, 0.1 mM phenylmethanesulfonyl fluoride Protease Inhibitor in 1 x PBS). Next, the homogenates were filtered through a 70 µm cell strainer to collect the nuclear fraction. The nuclear fraction was mixed with an equal volume of 50% iodixanol and added on top of a 30% and 33% iodixanol gradient. This solution was then centrifuged for 20 min at 10 000 ×g at 4 °C. After the myelin layer was removed from the top of the gradient, the nuclei were collected from the 30% and 33% iodixanol interface. The nuclei were resuspended in nuclear wash buffer and resuspension buffer and pelleted for 5 min at 500 ×g at 4 °C. The nuclei were filtered through a 40 µm cell strainer to remove cell debris and large clumps and the nuclear concentration was manually assessed using trypan blue counterstaining and a hemocytometer. Finally, the nuclei were adjusted to 700–1200 nuclei µL−1, and examined with a 10X Chromium platform. Reverse transcription, cDNA amplification and library preparation were performed based on the protocol from 10X Genomics. Briefly, nuclei were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 5' Reagent Kits v3.1. Barcoded, full-length cDNAs were then reverse-transcribed from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers which were used in Illumina bridge amplification. The Single Cell 5' Protocol produced Illumina-ready sequencing libraries. A Single cell 5' library comprised standard Illumina paired-end constructs which begin and end with P5 and P7. The Single Cell 5' 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1, while Read 2 was used to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 were standard Illumina® sequencing primer sites used in paired-end sequencing. Library sequencing was performed on the Illumina HiseqTM 4000 by Gene Denovo Biotechnology Co., Ltd (Guangzhou, China).