GSM4942425: Embryonic day E18.5 Hypothalamus, Control, f4530_E2S4; Mus musculus; RNA-Seq
The hypothalamus of control and Eed-cKO embryos was dissected out in ice-cold RPMI-1640 medium (cat.no. 11530586, Thermo Fisher Scientific, Waltham, MA, USA). The dissected hypothalamis were further divided into 2-4 pieces before cell dissociation for single cell isolation using Papain Dissociation System (cat no. LK003150, Worthington Biochemical Corporation, Lakewood, NJ, USA). The tissues were incubated with papain solution at +37°C for 60 min. Isolated single cells were re-suspended in ice-cold 1xPBS, 0.1% BSA, to a final concentration of 2,500 cells/μl (+/- 10%). Cells were checked for viability using a Bio-Rad TC10 automated cell counter (Bio-Rad, Hercules, CA, USA). Single cells and barcode beads were encapsulated into droplets using Bio-Rad ddSEQ Single-Cell Isolator for cell lysis and barcoding (cat.no. 12004336, Bio-Rad, Hercules, CA, USA). Subsequently, RNA-seq libraries were generated using Illumina SureCell WTA 3' Library Prep Kit for the ddSEQ System (6 cartridge version, cat.no. 20014280, Illumina, San Diego, CA, USA). Libraries were assessed for quality, using 1µl of undiluted cDNA, on an Agilent Technology 2100 Bioanalyzer using an Agilent High Sensitivity DNA chip (cat.no. 5067-4626, Agilent, Santa Clara, CA, USA) to determine fragment size and yield. Samples were stored at -20℃ until sequencing. Libraries were pooled according to their molar concentration (yield), fragment size, and the unique Nextera i7 indices (8bp). Each sequencing run consisted of four samples with different Nextera i7 indices. The libraries were sequenced on an Illumina NextSeq500 system, using NextSeq 500/550 High Output Kit v2.5 (150 Cycles, cat.no. 20024907, Illumina, San Diego, CA, USA), paired-end read type and Single-Index.
GEO
GSM4942425