title: GSM8069519: 12B_Unt_060320, NOMe; Homo sapiens; OTHER
library_construction_protocol: At 72 hours, viable (propidium iodide negative) single cells were isolated by Fluoresence activated cell sorting (FACS) and sorted into 2.5µL of RLT PLUS buffer (Qiagen) containing 2.5U SUPERas-In (Thermo Fisher Scientific) in LoBind 96-well full skirted plates (Eppendorf) using indexed sorting on a FACS Aria II (BD Biosciences). Plates were sealed and briefly centrifuged before storage at -80°C for sequencing analyses. For scNMT-seq, matched scNOMe-seq and scRNA-seq libraries were prepared from sorted HL-60 single cells, as previously described (Clark, S. J. et al. scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells. Nature Communications [2018]) with minor modifications described below and within the associated manuscript. Cell lysates were incubated with magnetic Dynabeads conjugated to the Bio-dT30VN oligonucleotide (5'- AAG CAG TGG TAT CAA CGC AGA GTA CT30VN-3'). Poly-adenylated mRNA captured on the Dynabeads underwent reverse transcription and cDNA amplification (for scRNA-seq), while genomic DNA was transferred to a new multi-well plate for bisulphite conversion (for scNOMe-seq). For reverse transcription, Dynabeads were resuspended in 10µL reaction mix containing: 100U SuperScript II reverse transcriptase (Thermofisher), 1X First-strand buffer (Thermofisher), 2.5mM DTT (Thermofisher), 1M Betaine (Sigma-Aldrich), 9mM MgCl2 (Thermofisher), 1μM Template switching oligo (5'AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3'), 1mM dNTPs (New England Biolabs) and 5U SUPERase-In RNAse inhibitor (Thermofisher). First-strand synthesis and template switching was then performed by incubating at 42 °C for 60 min, 50 °C for 30 min and 60 °C for 10 min. Amplification of cDNA was performed by adding KAPA HiFi HotStart ReadyMix (final concentration 1x, Roche sequencing) and ISPCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3', final concentration 0.11μM) to the reverse transcription mix and amplifying as follows: 98 °C for 3 min, 23 repeats of (98 °C for 20s, 67 °C for 15 s, 72 °C for 6 min) and 72 °C for 5 min. Amplified cDNA was stored at -20 °C for later scRNA-seq library preparation. Genomic DNA was purified using a 0.8x volume of AMPure XP beads (Beckman Coulter), and bisulphite converted using the Zymo EZ-96 DNA Methylation-Direct MagPrep Kit (Zymo Research) with halved reaction volumes. Converted DNA was eluted in first-strand synthesis mix containing: 1X Blue Buffer, 0.4mM dNTPs, 0.4 μM oligo1 - 5'CTACACGACGCTCTTCCGATCTNNNNNN 3'. Samples were incubated at 65 °C for 3 min and rapidly cooled before addition of 50 U of Klenow 3' -> 5' exo– (Enzymatics). The final reaction volume was 40µL, and samples were incubated as follows: 4°C 5 min, +1°C/15 s to 37°C, 37°C 30 min. Samples were heated to 95°C for 45s and rapidly cooled before addition of 2.5µL containing: 1X Blue Buffer, 0.4mM dNTPs, 0.4 μM oligo1 and 25U of Klenow 3' -> 5' exo–. The first strand synthesis step was then repeated (4°C 5 min, +1°C/15 s to 37°C, 37°C 30 min). In total, 5 rounds of first-strand synthesis were performed with the final round having an extended incubation for strand extension (4°C 5 min, +1°C/15 s to 37°C, 37°C 90 min). Samples were then incubated with 40 U exonuclease I (New England Bio-labs) for 1 h at 37 °C before DNA was purified using a 0.65x volume of AMPure XP beads. Samples were eluted in second-strand synthesis mix containing: 1X Blue Buffer, 0.4mM dNTP mix, 0.4 μM oligo2 (5'-TGCTGAACCGCTCTTCCGATCTNNNNNN-3'. Samples were incubated at 95°C for 45s and rapidly cooled before addition of 50 U of Klenow 3' -> 5' exo–. The final reaction volume was 40µL, and samples were incubated as follows: 4 °C for 5 min, +1 °C/15 s to 37 °C, 37 °C for 90 min. DNA purification was carried out using a 0.65x volume of AMPure XP bead buffer. Beads were resuspended in 1X KAPA HiFi HotStart ReadyMix with 0.2μM of i5 and i7 indexed primers from the NEBNext Multiplex Oligos for Illumina Dual Index Primer Sets (New England Biolabs, USA). Libraries were then amplified by PCR as follows: 95 °C 2 min, 12–14 repeats of (94 °C for 80 s, 65 °C for 30 s, 72 °C for 30 s), 72 °C for 3 min and 4 °C hold. Amplified libraries were purified using a 0.65x volume of AMPure XP beads and eluted in nuclease free water. Amplified cDNA samples were quantified using Qubit dsDNA HS Assay Kit (ThermoFisher Scientific) and normalised to 0.2 ng/μL with nuclease free water before proceeding with RNA-seq library preparation. The Nextera XT DNA library preparation kit (Illumina) was used according to the manufacturer's guidelines, but with halved reaction volumes and one in five dilutions of i5 and i7 indexed primers.