title: PARE of Arabidopsis rosette: RRGD Timepoint VII - recovery 30 min rep  1
design: RNA was isolated using Trizol Reagent (Life Technologies Australia Pty Ltd) using a procedure adapted from Allen et al. (2010). PARE libraries were prepared as per Zhai et al. (2014) with minor modifications included below. PARE libraries capture a 20-21 nt tag of the 5_ end of all uncapped (5_ monophosphate) mRNA molecules by ligation of a 5_ adapter incorporating the recognition site of the Type II restriction endonuclease MmeI. Libraries were prepared from polyA+ RNA captured from 75 lg of total RNA. Following ligation of the dsDNA 3_ adapter, the ligation product was PAGE purified to remove abundant adapter dimers, yielding higher quality final libraries. Following tag generation libraries were amplified with 3_ index primers from the Illumina Truseq sRNA Kit (RS-200-0012) and a custom PARE-TruSeq primer, facilitating incorporation into the Illumina workflow for sequencing. Pilot PCRs were employed to determine an optimal PCR cycle number of 8 cycles for the final amplification, which was performed as a half volume reaction (25 ll). Following the final PCR, the indexed libraries were pooled equal-molar then purified using AMPure beads using a bead ratio of 1:1.7 determined empirically using a standard curve to maximize recovery of the 130 bp library and minimize carryover of 80 bp contaminants. Libraries were sequenced on the Illumina HiSeq2500 in 50bp single end mode.