Metatranscriptomic sequencing of sediment RNA: Sample Core_39-10
"In order to get enough RNA for library preparation, RNA samples were amplified using the MessageAmp II-Bacteria Kit (Ambion) according to the manufacturer's protocol. The antisense RNA (aRNA) obtained was subjected to ribosomal RNA subtraction following the procedure of Stewart et al. (36) with the exception that the T7 promoter was coupled to the forward primer instead of the reverse primer. After subtraction, a 227 bp control RNA transcribed from the pSPT18 vector (positions 2867-3104 and 1-70) was added in a 1:1000 ratio (on a nanogram basis) to the total rRNA-subtracted RNA. This mixture was then reverse-transcribed using the SuperScript III kit (Invitrogen, Life Technologies). Illumina libraries were prepared following the protocol of Meyer and Kircher (30), with tags 1 to 34. The indexed libraries were pooled in an equimolar ratio and sent for eight lanes of Illumina HiSeq 2000 paired-end 2x100 bp sequencing at the McGill University and Genome Quebec Innovation Centre (Montreal, Canada)."
MG-Core_39-10