D. discoideum Lotte-seq rep1
Total RNA of D. discoideum cells were isolated using Trizol. The hairpin-shaped DNA adapter with 3'-TGGN overhang (5'-pCGACACTGTCGGTACCGACGGGAGAAGTCGGTACCGACAGTGTCGTGGNp-3') were ligated. tRNA ligated at the 3'-end to the hairpin adapter was incubated with 32P-labeled RT primer (5'-CAAGC TCGGTACCGACAGTG-3'). The SuperScript IV RT were added. The cDNA was separated on a poly-acrylamide gel. The DNA-only version of the Illumina TruSeq small RNA kit adapter (5'-pGATCGTCGGACTGTAGAACTCTGAAC-AminoC6-3') was ligated to the gel-purified cDNA. cDNA carrying 5' and 3' adapter sequences was incubated and amplified with the Illumina 5' PCR primer for TruSeq small RNA (5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3') and a custom index primer (5'-CAAGCAGAAGACGGCATACGAGATNNNNNNTAGGACTCCATGCAAGCTCGGTACCGACAGTG-3'). High-throughput analysis of the libraries was done as single end run (150 nt) on a Illumina MiSeq System with the custom primer (5'-CACTGTCGGTACCGAGCTTGCATGGAGTCCTA-3').