RNA-seq and RPF libraries were constructed exactly as described in Subtelny et al., 2014 (GSE52809) PAL-seq libraries were constructed and processed according to the methods. For RNA-seq or RPF libraries, detailed protocols are available (Subtelny et al., 2014 and Bartel lab website. NEXTflex libraries were prepared according to the manufacturer's instructions. PAL-seq library construction is as follows: Tail-length standard mixes (1 ng of set 1 and 2 ng of set 2 for each 5-EU-selected sample, and twice these amounts for the steady-state sample), and trace 5′-radoiolabeled marker RNAs (Table S2) were added to each sample to assess tail-length measurements and ligation outcomes, respectively. Polyadenylated ends including those with a terminal uridine were ligated to a 3′-biotinylated adapter DNA oligonucleotide (1.8µM) in the presence of two splint DNA oligonucleotides (1.25µM and 0.25µM for the U and A-containing splint oligos, respectively, Table S2) using T4 Rnl2 (NEB) in an overnight reaction at 18 °C. Following 3′-adapter ligation the RNA was extracted with phenol-chloroform (pH 8.0), precipitated, resuspended in 1X RNA T1 sequence buffer (ThemoFisher), heated to 50°C for 5 min and then put on ice. RNA was subsequently captured on streptavidin beads, 5′ phosphorylated, and ligated to a 5′ adapter as described (Subtelny et al., 2014) but using a modified 5′ adapter sequence (Table S2). Following reverse transcription using SuperScript III (Invitrogen) with a barcode-containing DNA primer, cDNA was purified as described (Subtelny et al., 2014), except a 160–810 nt size range was selected. Libraries were amplified by PCR for 8 cycles using Titanium Taq polymerase according to the manufacturer's protocol with a 1.5 min combined annealing/extension step at 57°C. PCR-amplified libraries were purified using AMPure beads (Agencourt, 40 µL beads per 50 µL PCR, two rounds of purification) according to the manufacturer's instructions.
GEO Accession
GSM3962917