GSM5618195: L. bicolor 5fC TOP-seq, replicate 1; Laccaria bicolor; OTHER
Mycelium was crushed to a powder with a pinch of polyvinylpyrrolidone (PVP, K25) using a mortar, pestle and liquid nitrogen. Powder (~1ml) was incubated at 55˚C for 90 min in 4.5 ml of lysis buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA, 2 M NaCl, 2% hexadecyltrimethylammonium bromide (CTAB), 2% PVP) with Proteinase K (Thermo Scientific, TS). Then, DNA was extracted using chloroform and precipitated for 30 min at 4˚C using 1.5 volumes of isopropanol and 0.4 volumes of 3 M sodium acetate pH 5.2. DNA was dissolved in 10 mM Tris-HCl and treated with RNase A/T1 (TS) for 1.5 h at 37˚C and then purified by chloroform extraction and ethanol precipitation. DNA quality was checked by agarose electrophoresis, concentration determined with Qubit™ dsDNA HS Assay Kit (Invitrogen™). For the hMe-Seal and fC-Seal libraries genomic DNA was sonicated on a Bioruptor UCD-200 (Diagenode) instrument in 10 mM Tris-HCl (pH 8.5) to on average 200-250 bp fragments and purified using DNA clean and concentrator kit (Zymo research, ZR).   5hmC protection and 5fC reduction to 5hmC was performed as in (Ličytė et al., 2020). To achieve similar processing of 5fC and 5hmC samples, the same purification steps were performed with both samples, but omitting glycosylation with unmodified UDP-glucose and reduction with NaBH4 for 5hmC. 2.6-2.7 μg of DNA was labeled using UDP-6-azide-glucose and T4 BGT (Thermo Scientific, TS) as described above (see Evaluation of 5hmC and 5fC enrichment). Two control DNA samples were prepared: for 5hmC control samples, BGT was omitted in the labeling step; for controlling 5hmC protection in 5fC samples, glucosylated DNA without the reduction step was used.   The biotinylation step was performed in a 130 μl reaction (as described in Evaluation of 5hmC and 5fC enrichment), and then, DNA was end-filled using a DNA End Repair Kit (TS) according to the vendor's recommendations and purified using DNA clean and concentrator kit (ZR). Biotinylated DNA was then enriched as described above in 40 μl using 0.1 mg of Dynabeads MyOne Streptavidin C1 (TS) and recovered by resuspending beads in 20 μl of 50 mM DTT, 60 mM Tris (pH 7.8), followed by a 1 h incubation on a roller. Released DNA was separated from the beads.  DNA was ligated overnight at 22 °C to 4.2 pmol of Ion Torrent™ barcoded adapters (Kapa Biosystems) in a 30 μl reaction containing 0.5 mM ATP, 10 mM MgCl2, 5% PEG 4000 and 7.5 U of T4 Ligase (TS). Adapter removal was then performed using MagJet NGS Cleanup and Size-selection kit (TS). Then, adaptor-ligated DNA was incubated with 10 mM 2-mercaptoethanol for 10 min at room temperature (to preclude inadvertent formation of inter-nucleotide disulphide cross-links), followed by PCR in 50 μl in Taq Buffer with (NH4)2SO4 supplemented with 0.2 mM dNTP, 2 mM MgCl2, 1 μM primers and 2.5 U of Taq DNA Polymerase (TS). PCR cycling conditions were: 1 min 50°C, 5 min 72°C, 4 min 94°C, 1 min 65°C, 5 min 72°C, 15 cycles of 1 min 94°C, 1 min 65°C, 1 min 72°C and the final extension step is at 72°C for 5 min. The libraries were size-selected for 300 bp fragments (MagJet NGS Cleanup and Size-selection kit, TS) and subjected to Ion Proton (TS) sequencing.  For hmTOP-seq and foTOP-seq libraries DNA was sonicated to yield ~200-250 bp fragments with Covaris M220 instrument and purified using DNA clean and concentrator kit (ZR). 5fC reduction to 5hmC, 5hmC protection, and azide-labeling of 5hmC (5.8 μg of DNA in this step) were performed as described above for the hMe-Seal and fC-Seal libraries. Then, DNA was end-filled for 15 min at 20°C using a DNA End Repair Kit (TS) according to the vendor's recommendations and purified using DNA clean and purified using the GeneJet Purification Kit. DNA was then dA-tailed in 120 μl with 30 u of Klenow exo- polymerase in its buffer (TS) in the presence of 0.5 mM dATP at 37°C for 45 min, enzyme inactivated at 75°C for 15 min followed by purification through GeneJet (TS) columns. Partially complementary annealed adaptors A1/A2 (2.25 μM, A1 5' P-GATTGGAAGAGTGGTTCAGCAGGAATGCTGAG and A2 5' ACACTCTTTCCCTACATGACACTCTTCCAATCT) were ligated in 120 µl by incubating the DNA with 30 u of T4 DNA Ligase (TS) in its buffer at 22°C overnight, followed by thermal inactivation at 65°C for 10 min and column purification (DNA Clean and Concentrator, ZR).  TOP-seq library enrichment and preparation was then done as in (Ličytė et al., 2020) and libraries were subjected to Ion Proton (TS) sequencing.