GSM7051139: single mES cell treated with ultrafast BS, replicate 1; Mus musculus; OTHER
Library preparation starting from mES genomic DNA: The mixture of mES gDNA (100-200 ng/µl) and spike-in lambda DNA (0.1%) were fragmented by sonication in 100 µl tube for 11 cycles of 30 seconds on and 30 seconds off using Bioruptor® Pico (Diagenode). The fragmented genomic DNA was size selected by Agencourt AMPure XP (Beckman Coulter) to 300-500 bp and eluted with 17 µl water. 3′-Dephosphorylation was conducted by adding 2 µl 10x T4PNK buffer (NEB) and 1 µl of T4 PNK (NEB) and the mixture was incubated at 37 °C for 30 min followed by 65 °C for 20 min. After purification by DNA Clean & Concentrate kit (Zymo Research), 164 bp dsDNA oligos (0.001%) was added as spike-in.  After BS treatment, all samples were desulphonated on Zymo-Spin IC columns following manufacturer's protocol except that final washing step was conducted for four times instead of twice. Then the samples were incubated at 95 °C for 5 min to denature DNA, and cold-shocked on ice for 2 min followed by libraries construction using xGen™ Methyl-Seq Lib Prep kit for 96rxn (IDT, catalog no: 10009824).   Library preparation starting from small number of mES cells (100-cell, 10-cell and single-cell): The DNA libraries starting from small number of mES cells were constructed following the previous method with slight modifications40,41. In brief, mES cells were sorted by fluorescence-activated cell sorting (FACS) into lysis buffer containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 20 mM KCl, 0.3% Triton-X 100, 1 mg/mL QIAGEN protease and 0.6 pg lambda DNA (dam-, dcm-). Genomic DNA was released under the program of 50 °C for 3 h, followed by 75 °C for 30 min. Bisulfite treatment was performed using UBS-1 and Zymo DNA Methylation-Gold kit side by side. The first-strand DNA was synthesized by random priming using 50 U Klenow exo-, 400 μM P5-N6-oligo1 (CTACACGACGCTCTTCCGATCTNNNNNN, IDT) at 37 °C for 30 min. After 4 rounds of random priming, samples were purified with 0.8x AMPure XP beads. The second strand was synthesized using P7-N6-oligo2 (AGACGTGTGCTCTTCCGATCTNNNNNN, IDT). After purification with 0.8x AMPure XP beads, the sequencing index was introduced by PCR using Multiplex Oligos for Illumina (NEB) and KAPA HiFi HotStart ReadyMix. Library preparation for human plasma cfDNA: 41 ng of plasma cfDNA either from healthy people or from patient's sample was spiked-in with 0.1% of the 164 bp dsDNA containing four 5mC sites and water was added to give each sample total volume 25 µl. Then to each sample, End Repair & A-Tailing Buffer (3.5 µl) and End Repair & A-Tailing Enzyme Mix from KAPA HyperPlus kit (Roche, catalog no: 07962428001) were added and the mixture was incubated at 65 °C for 30 min. Then a mixture of 15 µl ligation buffer, 5 µl DNA ligase, 2.5 µl water and 2.5 µl 15 µM methylated NEBNext adaptor was added, and the mixture was incubated at 20 °C for 1 h. Then 3 µl USER enzyme (NEB, catalog no: M5505S) was added and incubated at 37 °C for 15 min. Then the ligated DNA was purified by 1.0x Ampure beads and eluted with 41 µl water and divided into 5 parts. One part equivalent to 1 ng cfDNA will be directly amplified as input library. Two parts containing 10 ng cfDNA were treated with UBS condition followed by desulphonation the same procedure as described above for mES gDNA, and the other two parts equivalent to 10 ng cfDNA were treated with the conventional BS condition following manufacture's protocol. The purified DNA was then amplified using KAPA HiFi Uracil+ DNA polymerase (Roche, catalog no: 07959079001) and the libraries was purified by 0.7x Ampure beads.