GSM5197959: Curcumin Sample 2; Apis mellifera; Bisulfite-Seq
Phenol:Chloroform extraction (Rasmussen et al. 2016) Briefly, abdomen carcasses with adhering fat body were homogenized in ATL buffer (QIAGEN 19076) containing 0.4 mg Proteinase K (Sigma Aldrich P8044). Samples were then incubated at 56 °C with shaking at 400 rpm for 16 h in a Thermomixer (Eppendorf 5355 000.011). The lysate (500 µl) was extracted once in phenol:chloroform:isoamylalcohol (25:24:1) before the aqueous phase  was RNAse treated by incubating the sample with 5 µl of RNAse A (20 mg/ml) at 37 °C with shaking at 550 rpm for 30 min. The aqueous phase was extracted again using phenol:chloroform:isoamylalcohol (25:24:1)  before genomic DNA was precipitated by adding 1/10 volume equivalents of 3 M sodium acetate (pH 5.3),  10 µl of linear acrylamide (ThermoFisher Scientific AM9520) and 2.5 vol of ethanol, washed twice in 70 % ethanol, air dried, and dissolved in nuclease free water MethylC (Urich et al. 2015) Briefly, 2 µg of genomic DNA spiked with 10 ng of λ-phage DNA which is devoid of DNA methylation was fragmented using a Sartorius Labsonic M sonicator, with cycle settings at 0.5 and power/amplitude set to 30 %. Average fragment length was assessed on an Agilent Bioanalyzer DNA High Sense chip. Lower and upper size cut-offs were performed using Agencourt AMPure XP beads at a volume of 0.6x and 1.4x to DNA solution respectively. Sticky ends were repaired using Epicentre End-it DNA end repair kit according to the manufacturer's recommendations. Poly-Adenylated tails were added to blunt end fragments using New England Biolabs (NEB) Klenow Fragment (3'-->5' exo-) and NEBNext® dA-Tailing Reaction Buffer according to the manufacturer's recommendations. The reactions were cleaned up using Agencourt AMPure XP beads at a volume of 1.4x to DNA solution. Methylated adapters were ligated to eluted DNA using NEB T4 DNA ligase according to manufacturer's recommendations. Ligation reactions were purified twice using Agencourt AMPure XP beads at a volume of 1.0x to DNA solution. Eluted DNA was bisulfite converted using Human Genetic Signatures MethylEasy™ Xceed kit according to the manufacturer's recommendations. A low cycle PCR was performed to increase sequencing depth. To estimate the number of cycles needed, PCR reactions were quantified using KAPA Library Quantification Kit using the manufacturer's recommendations. Completed PCR products were cleaned up using Agencourt AMPure XP beads at a volume of 1.0x to DNA solution