GSM6606933
RNA was extracted using TRIzol according to the manufacturer's protocol. For library construction, 21μl DNA was mixed with 2μl of a universal i5 and a uniquely barcoded i7 primer, and then 25μl NEB Next HiFi 2× PCR Master mix was added and mixed. A total volume of 50μl sample was placed in a PCR thermal cycler with a heated lid via the following program: 72°C for 5min; 98°C for 30s; 16 cycles of 98°C for 10s and 63°C for 30s; final extension at 72°C for 1 min and hold at 4°C. To purify PCR products, 1.5 × volumes of AMPure XP beads (Beckman Coulter) were added and incubated for 15 min at RT. Next, the libraries were washed twice gently in 80% ethanol, and eluted in 30μl 10mM Tris pH 8.0. The size distribution of libraries was determined by Agilent 2100 TapeStation analysis.