Instrument: Illumina HiSeq 2500
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: PAIRED
Construction protocol: Sorted cells were lysed in 96 well plates in 4 uL of lysis buffer (20 mM Tris-HCl, pH 8.0, 20 mM KCl, 0.3% Triton-X 100, 1 mg/mL Qiagen Protease) for 30 minutes at 50 degrees. The lysed cells were then spiked-in with 1 uL of 60 fg/uL purified T7 phage DNA (GeneON). The DNA mixture was then bisulfite converted with the MethylEdge Bisulfite Conversion kit (Promega) using a modified bead-based protocol which enables automation (Domanico et al., 2013): 5 uL of the lysis/spike-in mixture was combined with 32.5 uL of MethylEdge Conversion reagent and incubated at 98°C for 8 minutes and 54°C for 60 minutes in a PCR machine. After incubation, the plate was briefly spun down and placed on an Agilent Bravo robot for subsequent steps. The 37.5 uL of bisulfite converted DNA was mixed with 180 uL of MethylEdge Binding Buffer and 1.8 uL of 20 mg/mL MagSi-DNA allround magnetic silica beads (MagnaMedics) and incubated at room temperature for 15 minutes. The beads were then collected on a magnet for 3 minutes and washed twice with 220 uL of 80% ethanol for 30 seconds while on the magnet. 60 uL of MethylEdge desulfonation buffer was added and incubated for 15 minutes. The desulfonation buffer was then removed and the beads were washed twice with 100 uL of 80% ethanol. The beads were then air-dried for 3 minutes, resuspended in 20 uL of Qiagen elution buffer, and incubated at 56°C in a shaking incubator at 2,000 rpm for 15 minutes. The beads were then collected on a magnet for 30 seconds and the supernatant containing the DNA was removed. The bisulfite-converted ssDNA from above was added directly into a mixture containing 1.25 uL of 10 mM dNTPs and 1 uL of 500 uM random hexamers (3’ phosphothioate). The reactions were incubated at 98°C for 1 minute and snap frozen on ice for 2 minutes. A mastermix of 0.5 uL of 50 U/uL Klenow fragment [3’→5’ exo-] (NEB M0212M), and 2.5 uL of 10X NEB Buffer 2 was added on ice and mixed well. The reactions incubated in a PCR machine as follows: 4˚C for 10 minutes, ramped from 4°C to 37°C at 4°C/s, and held at 37°C for 30 minutes. The entire reaction was denatured again at 98˚C for 1 minute and snap frozen on ice for 2 minutes. The reaction was transferred on a pre-chilled CoolRack by robot into a new plate containing 5 uL mixture of 20 uM random hexamers (3’ phosphothioate), 0.5 mM dNTPs, 1X NEB buffer and 25 U of Klenow fragment [3’→5’ exo-]. The wells were mixed well and the reaction was cycled once again: 4˚C for 10 minutes, ramped from 4°C to 37°C at 4°C/s, and held at 37°C for 30 minutes, and heat-inactivated at 70°C for 10 minutes. 20 uL of Qiagen Elution Buffer was added to each reaction to make it up to 50 uL, and each reaction was purified using 50 uL of Sera-Mag beads before proceeding to the standard illumina adapter ligation library construction protocol. The 25 uL of cleaned-up adapter-ligated fragments were PCR amplified in 50 uL containing 1 U of Phusion U Hot Start DNA Polymerase (Life Technologies F-555S), 1X HF buffer, 3% DMSO, 200 uM dNTPs, 0.5 uM PCR primer 1.0 (need sequence) (IDT), and corresponding 0.5 uM indexing PCR primer 2.0. The reactions were incubated in a PCR machine as follows: initial 98˚C for 1 minute; 10 cycles of 98˚C for 30 seconds, 65˚C for 15 seconds, 72˚C for 15 seconds; and a final extension of 72˚C for 5 minutes. The 50 uL reaction was cleaned up with 40 uL of Sera-Mag beads and eluted in 15 uL.