GSM4101821: Donor-blunt Acceptor-A; synthetic construct; OTHER
N/A; synthetic oligonucleotdes RNA sequencing libraries were prepared by using TGIRT-III (InGex), a commercial version of GsI-IIC RT, with different R2 RNA/R2R starter duplexes and acceptor nucleic acids as indicated in the text. The initial template template-switching reactions for addition of the R2R adapter to the 5' end of the cDNA were done as described above with 500 nM TGIRT-III enzyme, 50 nM unlabeled starter duplex, and 100 nM acceptor RNA for 15 min at 60 °C. After terminating the reactions with NaOH and neutralization with HCl as described above for template-switching reactions, the volume was raised to 100 μl with H2O, and cDNA products containing the R2R adapter attached to their 5' end were cleaned-up by using a MinElute column (Qiagen) to remove unused primer. A 5' adenylated R1R adapter was then ligated to the 3' end of the cDNA using Thermostable 5' AppDNA/RNA ligase (New England Biolabs) for 1 h at 65 °C. After another MinElute clean up, the entirety of the eluent was used for a 12 cycle PCR reaction using Phusion polymerase (ThermoFisher), and the resulting libraries were cleaned up by using 1.4x Ampure XP beads to remove residual primers, primer dimers, salts, and enzymes. The quality of the libraries was assessed by using a 2100 Bioanalzyer Instrument (Agilent) with a High Sensitivity DNA chip (Agilent). The libraries were sequenced on an Illumina NextSeq instrument to obtain 1-2 million 75-nt paired-end reads. Read 1 was used for analysis.
GEO Accession
GSM4101821