title: L597-gSMF236_1012_-dox-b2
design: Methylation treatment Between 1-3 million K562 cells were spun down (5 min, 300 r.c.f., 4C) in 2mL Eppendorf tubes in a pre-chilled swing-bucket centrifuge, which is used throughout the protocol. The supernatant was aspirated and cells were resuspended in 200uL ice cold PBS. After spinning cells down (5 min, 300 r.c.f., 4C), the supernatant was aspirated using a P200 to avoid cell loss. To lyse cells using the Omni-ATAC protocol, cells were then resuspended in 200uL of fresh ice cold lysis buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 minutes. After lysis, 1.2 mL of ice cold resuspension buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20) was added, and the tubes were inverted 3x to mix. Nuclei were then immediately spun down (10 min, 500 r.c.f., 4C). Supernatant was aspirated first with a P1000 and then with a P200 to avoid nuclei loss. Each methylation reaction was thoroughly mixed on ice using 100uL of cell methylation buffer (10% volume 10x M.CviPI reaction buffer (NEB, B0227S), 300 mM sucrose, 2.13 mM S-adenosylmethionine) and 50uL of 4,000U/mL M.CviPI GpC methyltransferase (NEB, M0227L). Each methylation reaction was then incubated for at 37 C shaking at 1000 rpm for 7.5 minutes (unless specified otherwise) and quenched with 150 uL gDNA cell lysis buffer, 3 uL RNase A and 1 uL ProtK all from the Monarch gDNA extraction kit (NEB, T3010). gDNA was extracted following the extraction kits protocol with the exception that 600 uL of gDNA binding buffer were added to 400 uL quenched reaction and added to the extraction column on two spins of 2 minutes at 1000g. DNA was quantified using Qubit 1x dsDNA HS Assay Kit on a Qubit Flex Fluorometer.   Enzymatic conversion  Methylated gDNA was digested using XbaI and AccI (NEB) to liberate a fragment containing the entire intact reporter sequence, and a 2-sided SPRI (0.4X followed by 1.8X) was performed to enrich for these fragments. DNA was converted using the Enzymatic Methyl-seq conversion module (NEB) following manufacturers instructions with slight modifications. First, 500 ng DNA (normally 200 ng) in 18 ul (normally 28 ul) was used as input to the oxidation reaction, where it was combined with 10 ul reconstituted TET2 reaction buffer + supplement, 1 ul oxidation supplement, 1 ul DTT, 3 ul oxidation enhancer (normally 1 ul) and 12 ul TET2 (normally 4 ul). This was mixed with 5 ul 0.4 mM Fe(II) solution and incubated for 1 hr at 37 C, after which 1 ul Stop reagent (ProtK) was added and incubated for 30 min at 37 C. 50 ul oxidized DNA was cleaned with 90 ul AmpureXP beads (1.8X), denatured by boiling in formamide at 90 C (normally 85 C) for 10 minutes, and quenched on ice. Deamination was performed by adding 10 ul APOBEC reaction buffer, 1 ul BSA, and 1 ul APOBEC, bringing the volume to 100 ul with H2O, and incubating at 37 C for 3 hr. 100 ul DNA was cleaned with 100 ul AmpureXP beads (1X) before library preparation.  Amplicon library preparation and sequencing  Amplicon library prep was performed in three rounds of PCR: 1) amplification out of the converted DNA 2) addition of part of R1 and R2 sequencing primers and 3) addition of remaining sequencing handles and sample indexes. For each 50 uL PCR1 reaction, we used 20 uL converted DNA, 2.5 uL each 10 uM forward and reverse primers (cTF223, cTF218 - see Supp. Table 2) and 25 uL NEBNext Q5U Master Mix (NEB, M0597) and ran the following thermocycling protocol: 30 seconds at 98 C, then 21 cycles of 98 C for 10 s, 61 C for 30 s, 65 C for 60 s, and then a final step of 65 C for 5 minutes. PCR 1 product was then cleaned with a 0.9x AmpureXP bead clean and eluted in 21 uL H2O. For each 20 uL PCR2 reaction, we used 1 uL cleaned PCR 1 product, 2.5 uL each 10 uM forward and reverse primers (cTF401/402/, cTF403/404 - see Supp. Table 2) and 10 uL NEBNext Ultra Q5 Master Mix and ran the following thermocycling protocol: 30 seconds at 98 C, then 6 cycles of 98 C for 10 s, 66 C for 30 s, 72 C for 45 s, and then a final step of 72 C for 5 minutes. Note that a mix of forward and reverse primers were used for PCR2, one set introduces an additional A after R1/R2 to improve sequence complexity. PCR 2 product was then cleaned with a 0.9x AmpureXP bead clean and eluted in 21 uL H2O. For each 25 uL PCR3 reaction, we used 20 uL cleaned PCR 2 product, 5 uL pooled 5 uM each forward and reverse indexing primers (oID1-7, oID8-24 - see Supp. Table 2) and 25 uL NEBNext Ultra Q5 Master Mix and ran the following thermocycling protocol: 30 seconds at 98 C, then 6 cycles of 98 C for 10 s, 68 C for 30 s, 72 C for 45 s, and then a final step of 72 C for 5 minutes. PCR 3 product was then cleaned with a 0.9x AmpureXP bead clean and eluted in 17 uL H2O. Libraries were then quantified using Qubit 1x dsDNA HS Assay Kit for concentration and D1000 ScreenTape (Agilent Technologies, 5067-5582) for length. Libraries were pooled with 15% PhiX Sequencing Control v3 (Illumina, FC-110-3001) and 15% genomic EM-seq libraries for complexity and sequenced with 374 cycles on R1, 235 cycles on R2, 8 cycles on I1, and 4 cycles on I2 and with a MiSeq Reagent Kit v3 (600-cycle) (Ilumina, MS-102-3003) on a MiSeq.