title: GSM5509981: Plate38_Lib1_scMAT-seq_H9; Homo sapiens; OTHER
library_construction_protocol: For single cell experiments, cells were sorted via FACs directly into lysis buffer.  For bulk experiments, genomic DNA was extracted using a Dneasy Blood & Tissue Kit from Qiagen. scMAT-seq  4 µL of Vapor-Lock (QIAGEN) was manually dispensed into each well of a 384-well plate using a 12-channel pipet. All additional dispenses into the 384-well plate were added using the Nanodrop II liquid handling robot (BioNex Solutions). To each well 100 nL of uniquely barcoded 7.5 ng/uL CEL-seq primer containing 4 UMIs was added. The CEL-seq type primers used here were described previously in detail (2). Then 100 nL of lysis buffer (0.175% IGEPAL CA-630, 1.75 mM dNTPs, 1:1,250,000 ERCC RNA spike-in mix (Ambion, 4456740), and 0.19 U RNase inhibitor (Clontech, 2313A)) was added. A single cell was isolated per reaction well using FACS. Unless cryopreserved, after sorting, plates were heated to 65 C for 3 min and returned to ice to lyse the cells. Next, 150 nL of reverse transcription GC tagging mix (0.7 U RNAseOUT (Invitrogen, 10777-019), 1.17x first strand buffer, 11.67 mM DTT, 3.5 U Superscript II (Invitrogen, 18064-071), 0.19 mM SAM, 1.17x GC reaction buffer, 0.1 U M.CviPI (NEB, M0227S)) was added and the plate was heated to 37°C for 1 hr, 4°C for 5 min, 65°C for 10 min, and 70°C for 10 min. Next, 1.75 µL of second strand synthesis mix (1.74x second strand buffer (Invitrogen, 10812-014), 0.35 mM dNTP, 0.14 U E.coli DNA Ligase (Invitrogen, 18052-019), 0.56 U E.coli DNA Polymerase I (Invitrogen, 18010-025), 0.03 U RNase H (Invitrogen, 18021-071)) was added and the plate was cooled to 16°C for 2hr. Afterwards, protease mix (6 µg Protease (Qiagen, 19155), 6.25x buffer 4) was added, and the plate was heated to 50°C for 15 hrs, 70°C for 20 mins, and 80°C for 5 mins. Next, 5hmCs were blocked by adding the following mix (1 U T4-BGT (NEB, M0357L), 6x UDP-glucose, 1x buffer 4) to each well and the plate was heated to 37°C for 16 hrs. Afterwards, protease mix (2 µg Protease, 1x buffer 4) was added, and the plate was heated to 50°C for 3 hrs, 70°C for 20 mins, and 80°C for 5 mins. Next MspJI digestion mix (1x buffer 4, 8x enzyme activator solution, 0.1 U MspJI (NEB, R0661L)) was added and the plate was heated to 37°C for 4.5 hrs, and 65°C for 25 mins. Unless otherwise stated, to each well 320 uL of uniquely barcoded 125 nM MspJI double stranded adapter was added. The MspJI adapters have been described in detail previously (3). Next 680 nL of ligation mix (1.47x T4 ligase reaction buffer, 5.88 mM ATP (NEB, P0756L), 140 U T4 DNA ligase (NEB, M0202M)) was then added, and the plate was cooled to 16°C for 16 hrs. After ligation, reaction wells receiving different barcodes were pooled using a multichannel pipette, and the oil phase was discarded. This was then incubated for 30 mins with 1x AMPure XP beads (Beckman Coulter, A63881), washing twice with 80% ethanol and resuspending in 30 µL of nuclease free water. Afterwards, this was evaporated down to 6.4 µL. Following this step, library preparation was performed as previously described in the scAba-seq and scMspJI-seq protocols (3, 4). Libraries were sequenced on an Illumina NextSeq 500 or an Illumina Hiseq 4000, sequencing a minimum of 75-bp on read 1 to detect 5mC. To detect RNA transcripts, a minimum sequencing length of 25-bp on read 1 and 50-bp on read 2 was utilized. Additionally, unless otherwise stated, transcript detection as only performed on RNA enriched samples and 5mC detection was only performed on unenriched samples.