title: GSM3619430: Medicago truncatula nodules harvested four days after watering withdrawal rep 1 [Mt.nodule.T4.1.S17]; Sinorhizobium meliloti; Medicago truncatula; RNA-Seq
library_construction_protocol: RNA was obtained from L. japonicus and M. truncatula non-inoculated roots and root nodules either subjected to drought for two (T2) or four (T4) days and control ones (T0). Total RNA extraction was performed using a GeneMATRIX Universal RNA Purification Kit (EURX, Gdańsk, Poland) with the additional step of on-column DNase I digestion. Per sample, an amount of 1 ug of total RNA was used as input. Using the Illumina TruSeq® Stranded Total RNA Sample Prep Kit (protocol 15031048 version E “October 2013”) rRNA is depleted from the total RNA samples using Ribo-Zero ribosomal RNA reduction chemistry. Illumina Ribo-Zero Plant (seed/root) kit was used for the plant samples, Illumina Ribo-Zero Gram negative bacteria kit was used for the bacterial samples and a mixture of both kits was used for the mixed samples. Subsequently, RNA was purified and fragmented and converted into first strand cDNA in a reverse transcription reaction using random primers. Next, double-stranded cDNA was generated in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. For each run, libraries were equimolar pooled and sequenced in two runs using high 75 cycles NextSeq v2 kits on an Illumina NextSeq 500 instrument.
attributes.tag: GEO Accession
attributes.value: GSM3619430