Illumina HiSeq 2500 sequencing
determination of transcription start sites
Approximately 2.5 ��g of DNase I treated RNA from the 4 sampling time points were mixed, and the rRNA in the RNA mixture was depleted using Ribo-Zero rRNA Removal Kit Bacteria (Epicentre). The rRNA-depleted RNA was incubated in 1�� RNA 5�� polyphosphatase (TAP; Epicentre) reaction buffer and 1 U of SUPERase-In (Invitrogen, Carlsbad, CA, USA) at 37 oC for 1 hr, with TAP. The reaction was cleaned up with ethanol precipitation and 5 pmol of 5�� RNA adaptor (5��-ACACUCUUUCCCUACACGACGCUCUUCCGAUCU-3��) was ligated to the purified RNA using T4 RNA ligase (Thermo Fisher Scientific, Waltham, MA, USA) by incubating at 37 oC for 90 min in 1�� RNA ligase buffer and 0.1 mg/mL BSA. The ligation product was then purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) according to the manufacturer��s instructions. The purified product was reverse-transcribed with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer��s instructions and purified using Agencourt AMPure XP beads. The purified cDNA was amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) for Illumina sequencing. The amplification step was monitored using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) and stopped before the PCR reaction was fully saturated. Finally, the amplified library was purified using Agencourt AMPure XP beads.