GSM8442847: WT_1; Amycolatopsis balhimycina; RNA-Seq
RNA was extracted using the “Zymo Quick RNA Fungal/Bacterial Kit” (Zymo Research, CN#R2014) according to the manufacturer's instructions. The RNA integrity was checked by agarose gel electrophoresis and residual DNA was removed by DNAseI treatment using the “DNA-free™ DNA Removal Kit” (ThermoFischer, CN#AM1906). Successful digestion was initially checked by PCR. The total RNA was quantified with a “Qubit RNA BR Assay Kit” (Thermo Fisher) and RNA integrity was checked by an Agilent 2100 BioAnalyzer with the “RNA 6000 Pico kit” (Agilent). Library preparation and ligation were performed using “Illumina Stranded Total RNA Prep” and “Ribo-Zero Plus Microbiome”, respectively, according to the manufacturer's instructions. In brief, 100 ng of total RNA per sample were subjected to rRNA depletion, followed by cDNA library construction, adapter ligation, and 15 cycles of barcoding PCR. The obtained libraries were quantified with the “Qubit 1x DNA HS Assay Kit” (Thermo Fisher) and the fragment distribution was checked with an Agilent 2100 BioAnalyzer using the “High Sensitivity DNA Kit” (Agilent). Libraries were subsequently pooled and sequenced on an Illumina NovaSeq 6000 device using “NovaSeq 6000 SP Reagent Kit” (v1.5) (100 cycles) with a run mode 75,10,10,0. The average number of reads obtained was 13-21x106. The sequencing was demultiplexed with the latest version of the nextflow pipeline: nf-core/demultiplex. For demultiplexing, “bcl2fastq” was used and the quality was checked with “fastp”.