T4 rna ligase truncated kq

The RACE protocol was performed as described (45 (link)). Briefly, total RNA was extracted using TRIzol (Ambion), followed by ligation of 5′-adenylated-3′-blocked adapter (New England Biolabs) using T4 RNA ligase truncated KQ (New England Biolabs). Ligated RNAs were purified and cDNAs were synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). PCR amplification was performed using gene specific primers and PCR amplicons were resolved on 2.5% agarose gels to visualize mature and extended transcripts. Primers used are given in Supplementary Table S1.

600 ng of RNA was ligated to 5 uM of 5′ adenylated, 3′ blocked adaptor (Universal miRNA cloning linker, NEB S1315S) with 250 units of T4 RNA ligase truncated KQ (NEB M0373S), 25% PEG 8000, and 1 μL RnaseOUT (ThermoFisher 10777019) in a 20 μL reaction at 25 degrees for 16 hours. Ligated RNA was cleaned up with RNA clean and concentrator columns (Clontech 740955.50) and DNase treatment, cDNA was synthesized with universal primer and SuperScript III (ThermoFisher 18080093). Amplification was carried out with Phusion (New England Biosystems M0530) and primer sets universal/TERCR1 (listed in the Key Resources Table). PCR products were directly run on an 8% PAGE gel and visualized with SYBR Gold (ThermoFisher S-11494), or subjected to AMPure XP beads (Beckman Coulter A63881) for PCR cleanup and library preparation. Libraries were prepped using Kapa Hyperprep Kit (Kapa KK8504), quantified with Qubit and bioanalyzer, and run on Illumina miSeq at the Stanford Functional Genomics Facility. Reads were paired using fastq-join tool at Galaxy (http://usegalaxy.org). Reads were binned into the various forms of hTR using custom python scripts (https://cmroake.people.stanford.edu/links-python-scripts) and the number of reads in each bin was normalized to total hTR reads.