title: GSM4142389: HUVEC infected with Ot Karp 1; Homo sapiens; RNA-Seq
library_construction_protocol: Both uninfected cells and infected cells were harvested by incubating the cells on ice and quickly resuspending in RNAprotect Bacteria Reagent (Qiagen, catalog no. 76506), then storing at -80 ¡C until use.  RNA extraction was performed using the Qiagen RNeasy Plus kit (Qiagen, catalog number 74136) according to manufacturer's instructions and as described previously (Atwal, 2017). The integrity of the DNase-treated RNA samples was assessed in a Bioanalyzer (Agilent). All samples had RIN (RNA integrity number) values ³8.0. Ribosomal transcripts were removed using the Ribo-Zero Gold (epidemiology) kit (Illumina). Following the manufacturer's instructions, 500 ng of total, DNase-treated RNA was used as an input to the ribo-depletion procedure. rRNA-depleted RNA was precipitated in ethanol for 3 h at -20¡C. cDNA libraries for Illumina sequencing were generated by Vertis Biotechnologie AG, Freising-Weihenstephan, Germany. rRNA-free RNA samples were first sheared via ultrasound sonication (four 30-s pulses at 4¡C) to generate on average 200- to 400-nt fragments. Fragments of 20 nt were removed using the Agencourt RNAClean XP kit (Beckman Coulter Genomics) and the Illumina TruSeq adapter was ligated to the 3' ends of the remaining fragments. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase (NEB) wherein the 3' adapter served as a primer. The first-strand cDNA was purified, and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10 to 20 ng/µl using a high fidelity DNA polymerase. The TruSeq barcode sequences were part of the 5' and 3' TruSeq sequencing adapters. The cDNA library was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis (Shimadzu MultiNA microchip).For sequencing, cDNA libraries were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200 to 600 bp using a differential cleanup with the Agencourt AMPure kit (Beckman Coulter Genomics). Aliquots of the cDNA pools were analyzed by capillary electrophoresis (Shimadzu MultiNA microchip). Sequencing was performed on a NextSeq 500 platform (Illumina) at Vertis Biotechnologie AG, Freising-Weihenstephan, Germany (single-end mode; 75 cycles).