title: GSM8070063: G03_AZA_220331, RNA; Homo sapiens; RNA-Seq
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 0.2μ