GSM7766363: Male genitalia, SpDAM+, Msx2Rtta+, Dox+, Tam+, biorep1; Mus musculus; OTHER
Freshly collected, never-frozen tissue followed by gentle enzymatic (non-mechanical) digestion is imperative to minimize background signal due to DNA breakage prior to DpnI methyl-specific blunt-end digestion. Freshly collected genital tubercles were subjected to digestion in 500uL PK lysis solution (100mM TrisCl pH 8.5, 5mM EDTA, 2% SDS, 20mM NaCl, with 0.5uL proteinase K per specimen, incubated at 52-55C overnight with shaking) followed by phenol-chloroform DNA extraction using Gel Lock Gel Heavy according to manufacturer's protocol. Following precipitation, resuspension, and treatment with RNaseA, 2.5ug gDNA per specimen was subjected to DpnI digestion (NEB cat. #R0176S) at 37C overnight in a total volume of 10uL and 20U DpnI enzyme per reaction, with heat inactivation at 80C for 20 minutes. In the same tube, samples were immediately subjected to bluntend ligation of double stranded adaptors (pre-annealed oligos 5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3' and 5'-TCCTCGGCCG-3' using T4 ligase (NEB cat. #M0202S) overnight at 16C with inactivation at 65C for 10 minutes. Immediately following adapter ligation, 3uL of DNA was subjected to adapter-specific PCR amplification using GoTaq-G2 polymerase (One 50uL RXN = 3uL adapter-ligated gDNA, 10uL 5x GoTaq buffer, 2uL 25x dNTPs, 1.25uL 50uM primer 5'phospho-GGTCGCGGCCGAGGATC-3', 33.5uL water, and 0.25uL GoTaq enzyme) under the cycling conditions pre-PCR 72C 10min  pre-PCR 94C 1min  pre-PCR 55C 5min  pre-PCR 72C 15min  6 cycles of 94C 1min followed by 55C 1min followed by 72C 10min,  25 cycles of 94C 1min followed by 55C 1min followed by 72C 2min  4C hold. The use of GoTaq-G2 negates the need for A-tailing prior to sequencing preparation due to the presence of single 3'-A overhangs in >80% of PCR products. At least 8 PCR reactions per specimen were pooled for PCR cleanup (Qiagen cat. #28104) to maximize library yield, with one additional reaction being used to run to agarose gel for quality control alongside untreated negative controls. Each DNA specimen was then prepared for sequencing by T4-ligation of pre-annealed Illumina index adapters (3uL at 50uM per 50uL RXN) in a reaction containing 5ug DNA according to manufacturer's protocol (NEB cat. #M0202S) overnight at 16C with inactivation at 65C for 10 minutes. Samples then underwent a second PCR cleanup, followed by size-exclusion by gel purification to avoid adapter dimers (Qiagen cat. #28704), followed by a third PCR cleanup to minimize buffer carryover. Samples then underwent an enrichment PCR prior to sequencing, again performing 8 or more reactions per sample to increase yield and pooling during purification. Phusion enrichment PCR (NEB cat. #M0530S) was performed using 250ng DNA per reaction and Y-adapter specific primers 5'- AATGATACGGCGACCACCGAGATCTACAC-3' and 5'-CAAGCAGAAGACGGCATACGAGAT-3' with cycling conditions 98C 30sec  20 cycles of 98C 40sec followed by 60C 30 sec followed by 72C 45sec  72C 5min  4C hold. This was followed by PCR cleanup, size-exclusion by gel extraction, and a final PCR-cleanup to minimize buffer carryover from gel extraction. Indexed samples were pooled at equimolar ratios for Illumina sequencing. OTHER: In vivo SpDAM seq