GSM8512151: HUG3_HGNP231, Human sperm, 3NP GAM; Homo sapiens; OTHER
A human germ cell sample was used for Genome Architecture Mapping (GAM) experiments, cells were fixed by 0.1% formaldehyde for 10 minutes while rotating at room temperature. Then, 2% BSA was added to quench formaldehyde. After being washed by Wash Buffer, the cell pellet obtained after centrifugation was used for ultrathin nuclear cryosection. Cryosections of 150 nm were cut with glass knives using a Leica FC7 ultra-cut cryotome, collected in sucrose droplets (2.3 M in PBS), and transferred to steel frame PEN (polyethylene naphthalate) membrane slides (Leica) for ultraviolet treatment for 45 minutes prior to use. Slides were washed in 1× PBS (three times, 5 minutes each), then with nuclease-free water (three times, 5 minutes each). Cresyl violet staining was performed with sterile-filtered cresyl violet (1 % w/v in water, Sigma-Aldrich, C5042) for 15 minutes, followed by two washes with water (30 s each) and air dried for at least 15 minutes. Prepare the lysis buffer (1× single-cell lysis buffer (refer to the recipe mentioned in Hi-C library generation), 6 mg/mL QIAGEN Protease ) in advance and add it to the 0.2 mL PCR tube caps (10 μL each) (Axygen). On average, three nuclear profiles were precisely microdissected using the Leica LMD7000 laser microdissection system and deposited into each 0.2 mL PCR tube cap. The tubes can be stored inverted at 4 °C for some days before whole genome amplification. Whole genome amplification of GAM sample DNA from microdissected nuclear profiles was performed with in-house protocol. In brief, NPs were lysed directly in the PCR caps for 4 hours at 60 °C. After protease inactivation at 75 °C for 30 min, the PCR tubes were inverted and centrifuged the right way up at 3,000 ×g for 5 minutes to collect the extracted DNA in the bottom of the tube. 10 μL of each lysed sample was transposed by the addition of 20 μL Transposition Mix (15 μL 2× Transposition buffer (20 mM 1 M Tris pH 8.0, 10 mM 1 M MgCl2, 16% w/v PEG8000), 0.2 μL TTE Mix V50 (dilute by a factor of 100) (Vazyme TD501), 4.8 μL water) and incubated at 55 °C for 10 minutes. Transposition was stopped by adding 1.5 μL SDS mix (0.9 μL 200mM EDTA, 0.6 uL 2% SDS) and then the tube was incubated at 55 °C for 10 minutes. Then, 1 μL 25 μM i5 unique dual (UD) index and 1 μL 25 μM i7 unique dual (UD) index were added in each tube. Next, the samples were amplified for 26 cycles of PCR with 16.5 μL KAPA PCR mix (Roche, KK2102) (10 μL 5× KAPA HiFi GC Buffer, 1.5 μL dNTP Mix (10 mM each), 2.5 μL 25mM MgCl2, 1 μL KAPA HiFi DNA Polymerase (1 U/μL) and 1.5 μL water). The barcoded nuclear profile libraries were then pooled and purified with 0.9× VAHTS DNA Clean Beads. The final libraries were sequenced with paired-end 150bp reads on a NovaSeq X plus (Illumina) platform according to the manufacturer's instructions.