Qiaprep spin miniprep kit

All primers were ordered from Invitrogen (Thermo Fisher Scientific). Synthetic DNA fragments were ordered either from Integrated DNA Technologies (IDT) or from Thermo Fisher Scientific. PCR and Gibson assembly were performed using Q5 hot start DNA polymerase and Gibson Assembly HiFi HC 1-step kit (SHI-DNA, Inc. CA), respectively according to the manufacturer’s directions. Plasmid DNA was isolated using the QIAprep spin miniprep kit and genomic DNA was extracted using Wizard genomic DNA purification kit (Promega). We used the Zymo clean gel DNA recovery kit to further purify DNA (Zymo Research).

Genomic DNA was bisulfite converted using the EpiTect bisulfite kit (Qiagen) and amplified in 50 μL PCR reactions containing 2 U ZymoTaq DNA polymerase (Zymo Research), 1x reaction buffer (Zymo Research), 250 μM each dNTPs, 1 μM forward primer, 1 μM reverse primer, and 50ng bisulfite converted DNA. Three primer pairs specific for the SSX2 promoter were used: forward 1: 5′-GGGTAGGGTGGTGTATGTTTGT-3′; reverse 1: 5′-ACCTTAACCAATCCTCCAACCT-3′; forward 2: 5′-GGAAGGATTTTTTGAGTTTAGGA-3′; reverse 2: 5′-TCTACCTTAACCAATCCTCCAA-3′; forward 3: 5′-AAGGATGATGGATTAATTAGGGT-3′; reverse 3: 5′-AATCCAAAAAAAAAATCAAACC-3′. Cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 30 sec, appropriate annealing temperature for 30 sec, 72°C for 1 min, and a final extension step of 72°C for 7 min. PCR products were purified using either the QIAquick PCR purification kit (Qiagen) or the QIAquick gel extraction kit (Qiagen). Purified products were then cloned into the pGEM-T easy vector (Promega), grown on AMP/IPTG/X-gal plates, cultured in suspension overnight, collected using the QIAprep spin miniprep kit, and sent to the UW-Madison Biotechnology Center for standard Sanger sequencing.