title: GSM5368903: scH&G_BseRI and AluI; Homo sapiens; OTHER
library_construction_protocol: H9 cells were grown on Matrigel (Fisher cat #08-774-552) in mTeSR1 (Stem Cell Technologies cat #85850). Cells were passed in clumps using Versene solution (Thermo fisher scientific cat # 15040066). For sorting, cells were dissociated into single cells using TrypLE, resuspended in 1x PBS, and passed through a cell strainer. 384-well plates containing 4 μL of Vapor-Lock (Qiagen) and 200 nL of lysis buffer (0.0875% IGEPAL CA-630) are prepared and single cells are FACS sorted into each reaction well. After sorting, plates are stored at -80°C until use. The cells were lysed at 65°C for 3 minutes, and reaction wells receive 500 nL of either 1.4x Buffer4 (NEB) [negative control, scAba-Seq only], BseRI mix [1.4x Buffer 4, 0.25 units BseRI (NEB)], AluI mix [1.4x Buffer 4, 0.25 units AluI (NEB)], or a combined mixture containing both BseRI and AluI (1.4x Buffer 4, 0.125 units BseRI, 0.125 units AluI). The plate is incubated for 1 hour at 37°C followed by heat inactivation at 80°C for 20 minutes. Next, 1.8 μL of protease mix (1x Buffer 4, 6 μg Qiagen protease) is added, and the plate is heated to 50°C for 16 hours, 75°C for 20 minutes, and 80°C for 5 minutes. Then 5hmC sites in the genome are glucosylated by adding 500 nL of glucosylation mix [1x Buffer 4, 1x UDP-Glucose (NEB), 1 unit T4-BGT (NEB)] and incubated at 37°C for 16 hours. Afterwards, 500 nL of protease mix (1x Buffer 4, 2 μg Qiagen protease) is added, and the plate is heated to 50°C for 3 hours, 75°C for 20 minutes, and 80°C for 5 minutes. To detect 5hmC, 500 nL of AbaSI reaction mix (1x Buffer 4, 1 unit AbaSI) is added and the plate is incubated at 25°C for 1.5 hours, and 65°C for 25 minutes. Cells receiving the AluI mix or the combined BseRI and AluI mix have 200 μL of 64 nM blunt end adapter added. All cell also receive 200 μL of 75 nM scAba-seq adapters. Ligation mix [1x T4 DNA Ligase reaction buffer (NEB), 4 mM ATP (NEB), 140 units T4 DNA Ligase (NEB)] is then added to bring the total volume of each reaction well to 5 μL. Subsequently, the plate is incubated at 16°C for 16 hours. Excluding the Vapor-Lock, all reagents are dispensed using the Nanodrop II liquid handling robot. After ligation, the reaction wells are pooled.