GSM9686060: Mouse colon tissue 3; Mus musculus; RNA-Seq
Mus musculus colon tissue were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 ml of ice-cold nuclei EZ lysis buffer. The sample was then incubated on ice for 5 min, with an additional 3 ml of cold EZ lysis buffer. Nuclei were centrifuged at 500g for 5 min at 4 C, washed with 5 ml ice-cold EZ lysis buffer and incubated on ice for 5 min. After centrifugation, the nucleus pellet was washed with 5 ml nuclei suspension buffer (NSB; consisting of 1 PBS, 0.01% BSA and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). Isolated nuclei were resuspended in 2 ml NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. The SC libraries were constructed according to the instructions of the DNA Nanoball (DNB) elab C4 scRNA Preparation Kit.  Briefly,  the cells were diluted to a concentration of 1,000 cells/mL and loaded into the cell reservoir of the microfluidic chip.  Barcoded beads  and droplet-generation oil were successively added to the beads and oil reservoirs.  Encapsulated droplets were generated and  collected using a DNBelab C4 system.  Beads that captured the mRNA were recovered for reverse transcription (RT).  Following  polymerase chain reaction (PCR) amplification by polymerase chain reaction, complementary DNA (cDNA) was purified and quantified using a Qubit dsDNA kit (Invitrogen, #Q32854).  Libraries of 30 -end transcripts were subsequently constructed through cDNA  fragmentation, size selection, end repair and A-tailing, adapter ligation, PCR for indexing libraries, and cyclisation of sequencing  libraries, according to the manufacturer's instructions.  Sequencing libraries were purified and quantified using the Qubit ssDNA  kit (Thermo Fisher Scientific, #Q10212).The sequencing library was performed on DNBSEQ-T7 platform(PE150) using DNBSEQ-T7RS Reagent Kit (FCL PE150) version 3.0.