QIAGEN Protease (30 AU)

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scMspJI-seq. Prior to FACS or manual isolation of single cells, 384-well plates (BioRad) are prepared as follows: 4 μL of Vapor-Lock (Qiagen) is manually added to each well using a multichannel pipette followed by 2 μL of lysis buffer (0.2 μL of 25 μg/μL Qiagen Protease, 0.2 μL of 10× NEB Buffer 4 and 1.6 μL of nuclease-free water) using the Nanodrop II liquid-handling robot (BioNex Solutions). All downstream dispensing steps are performed using the liquid-handling robot. After spinning down the 384-well plates, single cells are deposited into each well of the plate and incubated at 50 °C for 15 h, 75 °C for 20 min, and 80 °C for 5 min. 5hmC sites in the genome are then glucosylated to block downstream recognition by MspJI by dispensing 0.5 μL of the following reaction mixture: 0.1 μL of T4-BGT (NEB), 0.1 μL of UDP-Glucose (NEB), 0.05 μL of 10× NEB Buffer 4, and 0.25 μL of nuclease-free water. After incubation at 37 °C for 16 h, 0.5 μL the following reaction mixture is added: 0.1 μL of 25 μg/μL Qiagen Protease, 0.05 μL of 10× NEB Buffer 4, and 0.35 μL of nuclease-free water. The plate is then incubated at 50 °C for 5 h, 75 °C for 20 min, and 80 °C for 5 min. Thereafter, gDNA is digested by the restriction enzyme MspJI by the addition of 0.5 μL of the following reaction mixture: 0.02 μL of MspJI (NEB), 0.12 μL of 30× enzyme activator solution (NEB), 0.05 μL of 10× NEB Buffer 4, and 0.31 μL of nuclease-free water. The digestion is performed at 37 °C for 5 h followed by heat inactivation of MspJI at 65 °C for
20 min. Next, 0.2 μL of cell-specific double-stranded adapters are added to indi- vidual wells and these adapters are ligated to the fragmented gDNA molecules by adding 0.8 μL of the following reaction mixture: 0.07 μL of T4 DNA ligase (NEB), 0.1 μL of T4 DNA ligase buffer (NEB), 0.3 μL of 10 mM ATP (NEB), and 0.33 μL of nuclease-free water. The ligation is performed at 16 °C for 16 h. Next, wells con- taining unique cell-specific adapters are pooled using a multichannel pipette and incubated with 0.8× Agencourt Ampure (Beckman Coulter) beads for 30 min, washed twice with 80% ethanol and resuspended in 6.4 μL of nuclease-free water. Thereafter, in vitro transcription and Illumina library preparation is performed as described previously in the scAba-seq protocol

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