GSM1518947: 721_MAP_6h; Bos taurus; RNA-Seq
Total RNA was extracted from each individual sample and purified individually at 0, 2 and 6 hpi and used to prepare pooled strand-specific RNA-seq libraries as previously described by us (Nalpas et al., 2013). RNA was extracted using an RNeasy kit incorporating an on-column DNase treatment step (Qiagen Ltd., Crawley, UK) according to the manufacturer’s instructions. The quantity and quality of the RNA was assessed using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer with an RNA 6000 Nano LabChip kit (Agilent Technologies Ltd., Cork, Ireland). A260/280 ratios greater than 2.0 and RNA integrity numbers [RINs] greater than 8.5 were obtained for all total RNA samples purified across the infection time course. The protocol used for RNA-seq library preparation was adapted from a protocol previously published by our group (Nalpas et al., 2013). Thirty-five strand-specific Illumina® RNA-seq libraries were generated (seven libraries for the MAP-infected and control groups at each time point and seven 0 h time point control samples) using 150200 ng of total RNA. Samples were heated at 65°C for 5 min to disrupt RNA secondary structure and purification of poly(A)+ RNA was performed using the Dynabeads® mRNA DIRECT™ Micro Kit according to the manufacturer’s instructions (Invitrogen™/Life Technologies Ltd., Paisley, UK). Purified poly(A)+ RNA was then fragmented using 1× RNA Fragmentation Reagent (Ambion/Life Technologies Ltd., Warrington, UK) for 5 min at 70°C and precipitated using 68 mM sodium acetate pH 5.2 (Ambion), 227 ng/μl glycogen (Ambion) and 30 μl of 100% ethanol (Sigma-Aldrich Ltd., Dublin, Ireland). The RNA pellets obtained were then washed with 80% ethanol, air-dried for 10 min at room temperature and then re-suspended in 10.5 µl of DNase- and RNase-free molecular biology-grade H2O.  Synthesis of first strand cDNA was performed by incubating fragmented RNA with 261 mM Random Hexamer Primers (Invitrogen™), 1× first strand buffer (Invitrogen™); 10 mM DTT (Invitrogen™); 0.5 mM dNTPs; 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor; and 200 U SuperScript® II Reverse Transcriptase (Invitrogen™) using the following temperature profile: 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. First strand synthesis reaction mixtures were then purified using MicroSpin™ G-50 columns according to the manufacturer’s instructions (GE Healthcare UK Ltd., Buckinghamshire, UK).   Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich) and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™) and 50 U E. coli DNA Polymerase I (Invitrogen™). Reactions were incubated at 16°C for 2.5 h. The double stranded cDNA was subsequently purified using a QIAquick PCR Purification kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 µl of the provided elution buffer.  Blunt-end repair of cDNA samples was performed in 100 µl reaction volumes containing 1× T4 DNA ligase buffer with 10 mM dATP (New England Biolabs® Inc., Ipswich, MA, USA), 0.4 mM of each dNTP (Invitrogen™), 15 U T4 DNA polymerase (New England Biolabs®), 5 U DNA Polymerase I Large [Klenow] Fragment (New England Biolabs®) and 50 U T4 polynucleotide kinase (New England Biolabs®). Reactions were incubated at 20°C for 30 min and the cDNA was then purified using a QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions and eluted in 32 µl of the provided elution buffer.  Illumina RNA-seq adaptor ligation reactions (50 µl volumes) were performed using 21 µl of each of the phosphorylated blunt-ended cDNA (with 3'-dATP overhangs) samples and 1× Quick DNA ligase buffer (New England Biolabs®); 30 nM custom indexed single-read adaptors (Supplementary Table 1) and 15 U T4 DNA ligase (Invitrogen™). Reaction mixes were incubated at room temperature for 15 min and purified using a QIAquick MinElute Kit according to the manufacturer’s instructions (Qiagen) and eluted in 10 µl of the provided elution buffer. Adaptor-ligated cDNA was gel-purified using 2.5% agarose gels stained with 1 μg/ml ethidium bromide (Invitrogen™). Gels were electrophoresed at 100 V using 1× TAE buffer (Invitrogen™) for 75 min at room temperature. Size-fractionated bands corresponding to 200 bp (+50 bp) were excised from each sample and purified using a QIAquick Gel Extraction kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 µl of elution buffer. For generation of strand-specific RNA-seq libraries, the second strand of the gel-purified adapter-ligated cDNA containing uracil was enzymatically digested in 30 µl reaction volumes containing 1× Uracil-DNA Glycosylase buffer and 1 U Uracil-DNA Glycosylase (Bioline). These reactions were incubated at 37°C for 15 min followed by 94°C for 10 min.   PCR enrichment amplifications (25 µl) containing 9 μl of second strand-digested, adaptor-ligated cDNA; 1× Phusion® High-Fidelity DNA polymerase buffer (New England Biolabs); 334 nM each Illumina® PCR primer (Illumina® Inc., San Diego, CA, USA); 0.4 mM each of dATP, dCTP, DGTP and dTTP (Invitrogen™) and 1 U Phusion® High-Fidelity DNA polymerase (New England Biolabs®). PCR amplification reactions were performed with the following temperature cycling profile: 98°C initial denaturation for 30 s; 18 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 30 s; and 72°C final extension step for 5 min. PCR products were visualized following electrophoresis on a 2% agarose gel stained with ethidium bromide (0.6 µg/ml; Invitrogen™) and purified to remove PCR-generated adaptor-dimers using an Agencourt AMPure XP kit (Beckman Coulter Genomics, Danvers, MA, USA) according to the manufacturer’s instructions with final elution in 30 μl of 1× TE buffer.  All RNA-seq libraries were quantified using a Qubit® Fluorometer (Invitrogen™). RNA-seq library quality was assessed using an Agilent Bioanalyzer and Agilent High sensitivity DNA chip (Agilent) and confirmed that insert sizes were 200-250 bp for all individual libraries. Individual RNA-seq libraries were standardized and pooled in equimolar quantities (10 µM for each individual library). The quantity and quality of the final pooled library was assessed as described above prior to sequencing.  Cluster generation and sequencing of the pooled RNA-seq libraries was performed on an Illumina® HiSeq 2000 sequencer according to the manufacturer’s instructions.
GEO
GSM1518947
GEO Accession
GSM1518947