alias: GSM4413684
title: GSM4413684: AAP5, early plate, day 4, Replicate 1; Sphingomonas sp. AAP5; RNA-Seq
library_construction_protocol: For isolation and purification the RNeasy-kit (Qiagen) was used according to the manufacturer's manual. Modifications are listed below. Cells lysis was performed enzymatically using 15 mg/mL lysozyme in Tris-EDTA-buffer (ph 8.0) and mechanically by vortexing for 3 min with acid washed glass beads. A first digestion of genomic DNA was performed on the column, using RNase-free DNase I (Qiagen) according to the manufacturer's protocol. Total RNA was eluted in 88 µL RNase-free H2O and a second DNase I-digestion of genomic DNA was performed in solution, followed by a second RNeasy purification step. In this second purification an additional washing step with 80 % ethanol was performed prior to elution with 30µl RNase-free water. The concentration of the RNA was quantified using a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany) and the RNA integrity was assessed using Bioanalyzer 2100 (Agilent, Santa Clara, USA). Ribosomal RNA depletion:  The Ribo-Zero (gram-negative) kit to remove the prokaryotic rRNA, according to the manufacturer's instructions. Single end, strand specific cDNA libraries were prepared using the Scriptseq v2 RNA-Seq Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturers protocol. For sequencing equal volumes of libraries (12 pM) were multiplexed on a single lane. Sequencing was done on the HiSeq 2500 (Illumina, San Diego, CA, USA) using TruSeq SBS Kit v3 - HS (Illumina, San Diego, CA, USA) for 50 cycles resulting in 50 bp reads. Image analysis and base calling were performed using the Illumina pipeline v 1.8 (Illumina, San Diego, CA, USA).
identifiers.namespace: GEO
identifiers.id: GSM4413684