GSM6267686: Worker replicate 2 brain ATAC; Apis mellifera; ATAC-seq
Dissected brains were placed in 200 µl of PBS containing EDTA-free Complete protease inhibitors cocktail (Roche). Dissected brains were then gently homogenised by hand with a plastic pestle and centrifuged (900 × g / 5 min). Supernatants were removed and pellets resuspended in 1 ml of lysis buffer (10 mM Tris-HCl pH 7.5 / 10 mM NaCl / 3 mM MgCl2 / 0.2 % (v/v) IGEPAL CA-630 / EDTA-free Complete protease inhibitors cocktail) by pipetting for 30 seconds. 50 µl aliquots were removed, centrifuged (900 × g / 5 min) and pellets resuspended in 50 µl of tagmentation buffer (40 mM Tris-HCl pH 7.5 / 10 mM MgCl2 / 20 % (v/v) dimethylformamide) 0.75 µl of Tn5 transposase was added and tagmentation proceeded for 30 minutes at 37 ˚C. All transposed samples were purified using a DNA Clean & Concentrator-5 kit, using the binding buffer ratio for small PCR products (Zymo Research). Following purification, ATAC-seq library amplification was carried out with 2 × Q5 Polymerase Master Mix and 25 µM of Nextera primers. The optimal cycle number was determined using quantitative polymerase chain reaction (qPCR) as originally described.