TET2 KO U2OS Cells Rep 2
Sequences are from libraries prepared via bACE-seq (bisulfite-assisted APOBEC-coupled epigenetic sequencing) to map 5-hydroxymethylcytosine genome-wide. bACE-Seq libraries were made using the protocol from Fabyanic et.al. (PMID: 37640946) with minor modifications. In brief, genomic DNA was extracted using the Invitrogen PureLink genomic DNA mini Kit (Thermo Fisher Scientific, K182001) following the manufacturer's protocol. 83.2-93.4 ng of gDNA was used for each sample. To each sample, 1 µL of spike in control DNA (0.5 ng/µL) isolated from pCal7 plasmid expressing SssI is added to each sample. Every CpG is methylated in this control, which helps to assess bACE-seq conversion efficiencies. Samples were diluted to 10 µL in MilliQ water and pre-heated by incubating at 50oC for 20 minutes on a thermocycler with a heated lid set to 95oC. 32.5 µL of CT Conversion Reagent (Zymo EZ DNA Methylation Direct Kit, D5021) was then added to each sample before running the following program on a thermocycler (98oC for 8 min, 64oC for 105 min, followed by 98oC for 8 min, 64oC for 105 min), with the lid set to 95oC. Following the bisulfite reaction, the tubes were placed at -20oC for 16 hours or overnight. The bisulfite converted DNA was then purified using the Zymo EZ Methylation Direct Kit, eluting with 17 µL of MilliQ water for a final elution volume of 16 µL.   Each sample was incubated at 95oC for 3 minutes on a thermocycler, with the lid set to 105oC. The samples were then immediately placed into a dry-ice ethanol bath to snap cool them and were left in the cooling bath for 5 minutes. The samples were then placed on a pre-chilled PCR rack and kept on ice. 8 µL of premixed APOBEC reaction mixture (2.4 µL APOBEC reaction buffer (NEB, E7134A), 0.48 APOBEC (NEB, E7133AA), 0.48 µL BSA (NEB, B9000S), 4.64 µL water) was added to each tube and samples were incubated at 37oC on a thermocycler with the lid set to 95oC. After 30 min, the samples were mixed and placed back on the thermocycler for two and a half hours at 37oC with the lid set to 95oC. DNA was purified from the reaction using 2X Agencourt AMPure XP SPRI beads (add 2X volume of SPRI beads, incubate at room temperature for 10 minutes, place samples on magnet and wash 2 times with fresh 80% ethanol, dry for 3 min) and eluted in 9 µL Low EDTA TE (IDT).   Sequencing libraries were constructed using the xGen Adaptase Module with minor modification. 250 nM of random stubby index primer was added to each sample (xGen Adaptase Module, P5L_AD002_H). Samples were incubated at 95oC on a thermocycler with the lid set to 105oC for 3 min, then cooled on ice for 2 min. 10 µL of BST-based random priming mixture (2 µL 10X ISO AMP Buffer II (NEB, B0374S), 0.8 µL BST 3.0 (NEB, M0374S), 1.2 µL 200 mM MgCl2, 2.8 µL 10 mM dNTP, 3.2 µL water) was then added to each sample and incubated at 60oC for 1 hour on a thermocycler with the lid set to 95oC. Fragment ends were blunted and dephosphorylated by adding 2 µl Exonuclease I (NEB, M0293L) and 1 µl shrimp alkaline phosphatase (NEB, M0371S) to each sample. The samples were then incubated at 37oC for 30 min and purified using a 1.6X SPRI beads (add 1.6X volume of SPRI beads, incubate at room temperature for 10 minutes, place samples on magnet and wash 2 times with fresh 80% ethanol, dry for 3 min, elute) Samples were eluted in 10 µL Low EDTA TE (IDT). The samples were denatured by incubating at 95oC on a thermocycler with the lid set to 105oC for 3 min, then cooled on ice for 2 min. Samples were indexed and amplified using the PCR protocol in the xGen Adaptase Module using 21 cycles. The indexed samples were purified using 1.6X SPRI beads (add 1.6X volume of SPRI beads, incubate at room temperature for 10 minutes, place samples on magnet and wash 2 times with fresh 80% ethanol, dry for 3 min, elute) and eluted in 12 µL Low EDTA TE (IDT).  Libraries were sequenced using 2x150 paired end reads on an Illumina NovaSeq X Plus.