GSM7856899: HVAV1_reRNA_SF9_sRNAseq; Spodoptera frugiperda; RNA-Seq
The cell pellets were resuspended in 98% formamide and 10 mM EDTA and lysed at 70 °C for 10 min. The total RNA were then extracted from the lysate using Zymo Quick-RNA Miniprep Kit (Zymo Research) and used for small-RNA sequencing library preparation. Following on-column DNAse treatment, total RNA was eluted using water. ~200 ng of total RNA was used for rRNA depletion reactions using NEBNext rRNA kit (bacteria) following manufacturer protocols. For eukaryotic samples, custom probes designed using NEBNext Custom rRNA depletion tool were used. The rRNA depleted RNA was purified using a Zymo-Spin IIC column, treated with T4 PNK using T4 PNK buffer (NEB) at 37 C for 30 minutes before spiking in ATP and further incubating. Further end treatment was performed using mRNA Decapping Enzyme (NEB) pre-mixed with its buffer directly into the PNK reaction and incubating at 37 C for 30 minutes. End-repaired RNA was purified using a Zymo-Spin IIC column, and used for Collibri small-RNA-seq library preparation reactions following manufacturer protocols (INVITROGEN). The resulting NGS adapter ligated cDNA was then amplified with KAPA HiFi HotStart ReadyMix using NGS indexing primers for 25 cycles (ROCHE). Amplified DNA fragments between 150~500nt were size selected by gel extraction purification following gel-electrophoresis in a 4% SYBR-GOLD E-GEL. Before loading into the sequencer, library concentrations were quantified using KAPA-QUANT qPCR kit (ROCHE). Bulk RNA was subjected to rRNA depletion with NEBNext rRNA Kit for either bacteria or eukaryotes, depending on the biosample. The final library was prepared with the Collibri small-RNA-seq kit (INVITROGEN) according to manufacturer instructions.