cDNA circularization and library construction PCR

Purified cDNA was circularized with CircLigase ssDNA ligase (Lucigen) in 1X reaction buffer supplemented with 1 mM ATP, 50 mM MgCl₂, and 1M betaine for 3 hours at 60°C, followed by enzyme inactivation for 10 min at 80°C. One-fifth of circularized cDNA was directly used for library construction PCR with a common forward (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT^∗C-3′) and unique indexed reverse primers (5′-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT^∗G-3′, asterisks denote a phosphorothioate bond and NNNNNN corresponds to the reverse complement of an Illumina index sequence). Amplification was performed with KAPA HiFi DNA Polymerase (Roche) in 1X GC buffer with initial denaturation at 95°C for 3 min, followed by five to six cycles of 98°C for 20 s, 62°C for 30 s, 72°C for 30 s at a ramp rate of 3°C/sec. PCR products were purified with DNA Clean&Concentrator 5 (Zymo Research) and resolved on 8% polyacrylamide/1XTBE gels alongside pBR322 DNA-MspI Digest (NEB). The 130-220 bp region of each lane was excised and DNA was eluted from crushed gel slices in 400 μl water with continuous mixing at room temperature overnight. After ethanol precipitation in 0.3M sodium acetate pH = 5.5 and 25 μg glycogen, libraries were dissolved in 10 μl 10 mM Tris-HCl pH = 8.0, quantified with the Qubit dsDNA HS kit, and sequenced for 150 cycles on an Illumina NextSeq platform.