title: GSM8210455: satcutaneous adipose tissue, replicate 1, scRNAseq; Bos taurus; RNA-Seq
library_construction_protocol: The approximately 100mg liquid nitrogen frozen tissue was ground onto ice and 2 mL of pre-cooled Homogeniza Buffer was added, which was put for 3-5 minutes until the cell nucleus were fully released under the microscope. The tissue homogenate was filtered using 70 µ m and 40 µ m filters, and centrifugated at 500g at 4 ℃ for 5 minutes. Discarding the supernatant and washing twice with 1 mL Wash Buffer, the precipitate was resuspended with an appropriate volume of Blocking Buffer, mixed well and observed the background in the nucleus suspension under the microscope. If the suspension background was clean, it could be directly counted and the cellular ornuclei suspensionwasstainedwith 0.4% trypan blue to assess cell viability under microscope. Cells with greater than 80% viability were qualified for library construction process. The single-cell suspension was partitioned into GEMs (Gel Beads in Emulsions) in the automated Chromium Controller, and then mRNAs were reversely transcribed into cDNAs, configured for breaking GEMs, which were purified for PCR. The cDNAs were subjected to fragmentation, end repair and addition of “A” base at the 3'-end of each strand, which were subjected to adaptor ligation and PCR. Basing on the requirements of good library quality, single-stranded PCR products were produced via denaturation and then circularized to get single-stranded cyclized products. The uncyclized linear DNA molecules were digested. Single-stranded circle DNA molecules were replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contains multiple copies of DNA was generated.