Transcriptome profiling of the mouse suprachiasmatic nucleus at single cell resolution

Circadian rhythms are governed by the hypothalamic suprachiasmatic nucleus (SCN) which consists of ca. 20,000 cellular oscillators bound together in a powerful time-keeping network. The goal of this study was to characterise its component cells and network structure by conducting single-cell sequencing of SCN organotypic slices. scRNASeq libraries were prepared using 10x Genomics Chromium Single Cell 3' Library and Gel Bead v2 kit. SCN cells, diluted in 0.04% BSA-nuclease-free water, reverse transcription master mix and partitioning oil, were loaded on a ChromiumTM Single Cell A Chip and run on the Chromium Controller to obtain at target cell recovery rate of 8,000 cells. Final libraries were sequenced on one flow cell of an Illumina HiSeq 4000 with a read length of 26bp for read 1 (cell barcode and unique molecule identifier and sample barcode) and 98bp for read 2 (RNA read) to yield approximately 340 million reads per sample. We sequenced 13,324 cells from 52 SCN slices at circadian time 7.5 (mid-day) over three independent sequencing runs, and 16,996 cells from 36 SCN slices at circadian time 15.5 (mid-night) over two independent sequencing runs. 10X Genomics Single Cell Transcriptome Sequencing of the mouse SCN小鼠下丘脑视交叉上核的10x Genomics单细胞转录组测序 昼夜节律（circadian rhythms）由下丘脑视交叉上核（hypothalamic suprachiasmatic nucleus, SCN）调控，该核团由约2万个细胞振荡器组成，彼此联结为功能强大的计时网络。本研究旨在通过对SCN器官型脑片开展单细胞测序，解析其组成细胞与网络结构。 本研究采用10x Genomics Chromium单细胞3'文库及磁珠v2试剂盒构建单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）文库。将以0.04%无核酸酶牛血清白蛋白（bovine serum albumin, BSA）水溶液、反转录预混液及分区油稀释的SCN细胞上样至Chromium™单细胞A芯片，并在Chromium控制器上完成运行，以达到8000个细胞的目标细胞回收率。最终文库在Illumina HiSeq 4000的单个流动槽上进行测序：读长1（read 1）为26bp，用于读取细胞条形码、唯一分子标识符及样本条形码；读长2（read 2）为98bp，用于RNA序列读取，每个样本可产生约3.4亿条测序读段。 本研究通过3次独立测序，对昼夜节律时间7.5（正午）的52个SCN脑片中的13324个细胞完成测序；同时通过2次独立测序，对昼夜节律时间15.5（午夜）的36个SCN脑片中的16996个细胞完成测序。