GSM4475233: SADRT39; leaf litter metagenome; RNA-Seq
We carried out RNA extraction on a coarse-ground litter aliquot of 0.2 g for shrub and 0.5 g for grass using RNeasy PowerSoil Total RNA Kit following manufacturer instructions (Qiagen, Hilden, Germany). Due to a high amount of organic compounds co-extracted from shrub litter, a lower amount of starting material was used in the extraction protocol to increase the RNA yield. After resuspending the RNA pellet in solution SR7 in the final step, purity and concentration of total RNA was assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), Qubit fluorometer (LifeTechnologies, Carlsbad, CA, USA) and Nanodrop 2000 Spectrophotometer (Thermo Scientific, USA). Ribosomal RNA was removed using a Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions with a modification that included combining magnetic beads from the yeast and bacteria kit as follows: 0.5x Yeast Removal Solution, 0.25x Gram Negative Bacteria Removal solution, and 0.25x Gram Positive Bacteria Removal Solution. Strand-specific and barcode indexed RNA-seq libraries were then generated using the Kapa RNA-seq Hyper kit (Kapa Biosystems, Cape Town, South Africa) following the instructions of the manufacturer. The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a Bioanalyzer 2100. The libraries were quantified by fluorometry on a Qubit fluorometer and pooled in equimolar ratios. The pool was quantified by qPCR with a Kapa Library Quant kit (Kapa Biosystems). The pooled library was sequenced on 1 lane of an Illumina HiSeq 4000 (Illumina, San Diego, CA, USA) with single-end 100 bp reads. The sequencing was carried out at the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center.