Metagenomic RNA sequencing of bat guano
cDNA was synthesized through priming the first and second strands with random hexamers. SuperScript IV (SSIV) Reverse Transcriptase (RT) kit was used for first strand synthesis. To obtain the 5 end of the sequence, 5 RACE template switching was performed with a Template specific oligo using the recommended protocol from New England Biolabs (NEB): 5 RACE with the NEB template switching RT Enzyme mix. We used the recommended template specific oligo and designed an upstream 5 cDNA GSP. As 5 RACE products did not yield a single clear amplicon, an unfragmented library was prepared using KAPA Biosystems Library Preparation kit using Truseq DNA barcodes. The libraries were sequenced on an Illumina Novaseq with a read length of 100 bp.