GSM5221561: Control sample – Replicate 3; Stylophora pistillata; RNA-Seq
Samples were flash frozen in liquid nitrogen in WhirlPack bags, to be crushed in small pieces with a press. Fragments were put in 600uL of lysis buffer RLT in PowerBeads Glass 0.1mm (Qiagen), samples were lysed in CryoMill bead beating instrument (Retsch) for 4 minutes with a program of 30 cycles/s. Samples were spin at max speed for 3 minutes and the 600uL of supernatant was transferred to a fresh tube. A 600uL (1V) volume of cold EtOH 70% was added to the column (Qiagen, RNeasy Mini Kit), gently mixed by pipetting, and immediately applied on the column to be then centrifugde 15s at 8 000g (10 000 rpm). Flow-through was discarded and the step was repeated. A 350 uL volume of RWI buffer was applied to the column and centrifuge 15s at 8 000g (10 000 rpm). Flow-through was discarded and 70ul of RDD buffer was added to 10uL DNAse, Rnase free, (Qiagen, 79254) stored at -20°C, to be then applied to the column. Leave the column at RT for 15min before adding 350 uL of RWI buffer and centrifuge 15s at 8 000g (10 000 rpm), discard the flow-through and add 500uL of RPE buffer, repeat the centrifugation, add again 500uL of RPE buffer and make a 2min at 8 000g (10 000 rpm) centrifuge before discarding the flow-through and putting the column in a clean 1.5ml tube. Add 30ul of RNase free water to the column and centrifuge for 1min at 8 000g (10 000 rpm), keep the tube on ice for further experiment and store at -80°C Libraries were prepared according to standards Illumina's instructions