Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: At 1, 4 and 24 hours post-infection, infected macrophages were washed twice with PBS and homogenized by adding Trizol reagent (Sigma). All the aliquots were stored at -80°C until required for RNA extraction. RNA extraction was performed by adding 200uL of chloroform (Sigma) to each sample and mixing by vortexing for 60 sec. The solution was then centrifuged for 15 min at 12000xg at 4°C. The aqueous phase was removed, transferred to a separate tube containing 500uL of isopropanol (Sigma), gently mixed and centrifuged 10 min at 12000xg at 4°C. The supernatant was discarded, the RNA pellet was resuspended in 1mL of 75% Ethanol, mixed by vortexing and centrifuged 5 min at 7500xg at 4°C. After discarding all the supernatants, the RNA was transferred to a miRNeasy mini kit column (Qiagen, Ldt.) and further purification steps were conducted according to the manufacturer’s instructions. In addition, the RNA was subjected to an on-column DNase treatment step (Qiagen Ltd.) to remove any DNA residues that could affect the downstream reaction. RNA quantity and quality was assessed using a NanoDrop TM 1000 spectrophotometer (Thermo Fisher Scientific Inc.) and on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Ltd.). All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers (RIN) greater than 7,5. Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit according to the manufacturer’s protocol. Briefly, the poly-A containing mRNA molecules were purified from 4ug of total RNA (extracted from not-infected and infected samples not bead-beaten) using poly-T oligo attached magnetic beads. The mRNA was then fragmented (to approximately 300bp) and the first strand cDNA was synthesized using reverse transcriptase (SuperScript II, Invitrogen) and random hexamers. The second strand of the cDNA was done by removing the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate a ds cDNA. dsDNA was then subjected to the addition of “A” bases to 3’ ends and ligation of the barcoded TruSeq adapters. Amplification of the fragments that had adapters ligated on both ends was performed by PCR using the primer cocktail supplied in the kit. Final libraries were analysed on an Agilent 2100 Bioanalyzer using the Agilent DNA 1000 chip (Agilent technology) to confirm that fragment size was ~200-260bp. All libraries were then quantified using a Qubit fluorometer and Qubit double stranded DNA High Sensitivity kit (Invitrogen). Libraries were loaded at a concentration of 10pM onto the flowcell and were single-end sequenced on Illumina HiSeq 2000 or Illumina MiSeq.