HIT-scISOseqV2 of mouse spermary cells
The dissociated single cells were processed through the GemCode Single Cell Platform per manufacturers recommendations using the Chromium Single Cell 3 GEM, Library, and Gel Bead Kit v3 (10x Genomics; PN-1000075). In V2 version, we used exonuclease I to treat the solution after emulsification. In the template switching, we used the different template switching primer AAGCAGTGGTATCAACGCAGAGTACrGrG+G. cDNA products were amplified with KAPA HiFi HotStart Uracil 2 x ReadyMix (Kapa Biosystems) and newly designed PCR primers that contain a deoxyuraciland one (5 different primer sets for 5 PCR reactions, in order to create 1-2-3-4-5 sequential ligations). The pooled PCR products from 5 PCR reactions were digested by USER Enzyme (NEB) and ligated together by T4 DNA ligase and processed to the Pacbio sequencing.