Terminome-Seq of Arabidopsis thaliana 21 days old leaves
All libraries were produced from 1ug of DNase I-treated RNA (www.neb.com), and for TAP-treated samples, tobacco acid phosphorylase (TAP; www.epibio.com) was used according to the manufacturer's instructions with heat inactivation at the end of the incubation period. Library synthesis was carried out using the Illumina TruSeq Small RNA library preparation kit (www.illumina.com) intended to capture the RNA population containing a 5' phosphate and 3' hydroxyl group. Minor modifications were made to the protocol depending on whether a native 5' or 3' end was the target. Libraries intended for native 3' end capture followed the protocol with initial 3' adapter ligation using T4 RNA ligase 2, a deletion mutant that can only ligate a 3' hydroxyl group to a 5' adenylated RNA, consistent with the 3' RNA adapter chemistry. After ligation, the RNA was fragmented using a Covaris sonicator (www.covaris.com), with a target size of 200 nt, followed by ethanol precipitation for concentration and 5' adapter ligation with T4 RNA ligase. Libraries intended for native 5' end capture required further adjustments. The order of adapter ligation was reversed: 5' adapter ligation (with T4 RNA ligase) – sonication – ethanol precipitation – 3' adapter ligation (with T4 RNA ligase 2). In this case, excess 5' adapter remaining following the sonication and ethanol precipitation could ligate to added 3' adapter, but not to any new 5' ends created through sonication as the new 5' ends would not be adenylated. This resulted in unwanted adapter dimers that were preferentially amplified during library amplification (PCR1) due to their small size (~133 bp). Therefore, size selection was performed on the products from PCR1, retaining only products over 200 bp using Pippin Prep (www.sagescience.com). A second PCR amplification (PCR2) was executed on these products. Quality control was performed after Pippin size selection and before library submission for sequencing using an Agilent BioAnalyzer (www.agilent.com). Details about the procedure and the Pippin Prep are available at https://github.com/BenoitCastandet/Terminome_Seq. The final cDNA libraries were purified using magnetic AMPure beads (www.beckman.com) following the manufacturer's protocol. Multiple steps in the above protocol, including fragmentation, ethanol precipitation, Pippin size selection (5' libraries only), and AMPure purification of cDNA libraries, resulted in a bias towards the retention, and therefore sequencing, of fragments >67 nt. Libraries were pooled and sequenced on a NextSeq500 Sequencer (www.illumina.com) using the v3 kit, with paired-end reads generating 40 bp long R1 reads and 35 bp long R2 reads for all libraries. R1 reads are only of use for libraries generated to obtain 5' related data, while R2 reads contain data related to 3' ends and therefore are only relevant for libraries generated to obtain 3' data.