FLOUR-seq of mouse spermary cells
The dissociated single cells were processed using the BD ExpressTM Platform, following some of the manufacturer's recommendations. This included performing a Single cell 3 whole transcriptome amplification (WTA) with sample multiplexing kit (SMK) and AbSeq protocol, using the BD RhapsodyTM Cartridge Reagent Kit (633731, BD biosciences), BD RhapsodyTM Cartridge Kit (633733, BD biosciences), and BD RhapsodyTM cDNA Kit (633773, BD biosciences).To elaborate, 20,000 dissociated cells and Cell Capture Beads were loaded into a pre-treated BD Rhapsody Cartridge, followed by lysing the cells with lysis buffer. The beads were washed with Bead Wash Buffer, and reverse transcription was performed. The tube containing the beads was placed on a magnet, and the 200 L of suspension was moved to a new tube and saved for the following template switching reaction. Finally, the beads were treated with exonuclease I.The beads treated with exonuclease I were resuspended in the saved 200 L reverse transcription reaction mix, supplemented with 5 L of 100 M template switching oligo (AAGCAGTGGTATCAACGCAGAGTACrGrG+G) and 2 L of 1 M MgCl2. They were then incubated on the thermomixer for 30 minutes at 1,200 rpm and 42C. The tube was placed on a magnet and the supernatant was removed. The beads containing cDNA were suspended in 200 L of PCR reaction mix, comprised of 100 L of KAPA HiFi HotStart 2x ReadyMix (Kapa Biosystems), 10 L of PCR primer 1 (5-GCTGTACCGATGCTAGGCTGTTCGTCACCATAGTTGCGTCTCATG-3), 10 L of PCR primer 2 (5-AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3), and 80 L of nuclease-free water. The beads were fully resuspended, and 25 L of PCR reaction mix with beads was pipetted into each of eight 0.2-mL PCR tubes. The PCR reaction was performed in a PCR thermal cycler for 12 cycles, followed by purification with 0.8x Agencourt AMPure XP Beads (Beckman Coulter).The amplified PCR products underwent end-repair, A-tailing, and ligation with a sequencing adapter using the Ligation Sequencing Kit (SQK-LSK110, Oxford Nanopore Technologies). The library was then sequenced on the Oxford Nanopore PromethION sequencing platform.