title: GSM1468846: 39_infected; Bos taurus; RNA-Seq
library_construction_protocol: RNA extraction was performed within two hours of collection, The cell pellet was fully resuspended in 2ml Trizol® reagent (Invitrogen Ltd., Paisley, UK) and RNA was extracted as per the manufacturer’s instructions.  RNA quantity and quality was assessed using both the NanoDropTM 1000 spectrophotometer and the Agilent 2100 Bioanalyzer In total, 18 strand-specific RNA libraries for high-throughput sequencing were prepared (eight libraries for each group: M. bovis-infected and control samples) using 1 μg of total RNA. Total RNA was first heated at 65°C for 5 min to disrupt any secondary structure. Purification of poly(A) RNA was performed using a Dynabeads® mRNA DIRECTTM 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 Corporation, 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). Pellets were washed with 80% ethanol, air-dried for 10 min at room temperature and re-suspended in 10.5 µl DNase- and RNase-free water. 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 RNaseOUTTM Recombinant Ribonuclease Inhibitor; and 200 U SuperScript® II Reverse Transcriptase (Invitrogen) at 25°C for 10 min, at 42°C for 50 min, and 70°C for 15 min. First strand synthesis reaction mixtures were purified using MicroSpinTM G-50 columns according to the manufacturer’s instructions (GE Healthcare UK Ltd., Little Chalfont, 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 mix 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 was performed in a 100 µl reaction containing 1× T4 DNA ligase buffer with 10 mM dATP (New England Biolabs® Inc., 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. To facilitate Illumina® GA adaptor ligation, a single ‘A’ base was added to the 3' ends of the blunt-end repaired cDNA samples. 32 µl of purified phosphorylated blunt end-repaired cDNA was included in a final 50 µl reaction mixture containing: 1× Klenow fragment buffer (New England Biolabs); 0.2 mM dATP (Invitrogen), and 15 U Klenow fragment with 3'-to-5' exonuclease activity (New England Biolabs). Reactions were incubated at 37°C for 30 min, after which cDNA was purified using a QIAquick MinElute Kit (Qiagen) according to the manufacturer’s instructions and eluted in 21 µl of the provided elution buffer. Illumina® RNA-seq adaptor ligation reactions (50 µl volumes) involved incubation of 21 µl of phosphorylated blunt-ended cDNA containing a 3'-dATP overhang with 1× Quick DNA ligase buffer (New England Biolabs); 30 nM custom indexed single-read adaptors (see Samples_barcode.xlsx) 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 Volts 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. To generate strand-specific RNA-seq libraries, the second strand of the gel-purified adapter-ligated cDNA containing uracil was digested enzymatically in 30 µl reaction volumes containing 1× Uracil-DNA Glycosylase buffer and 1 U Uracil-DNA Glycosylase (Bioline). Reactions were incubated at 37°C for 15 min followed by 94°C for 10 min. PCR enrichment amplifications (50 µl) containing 15 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 consisted of an initial denaturation step of 98°C for 30 seconds, 18 cycles of 98°C for 10 seconds, 65°C for 30 seconds and 72°C for 30 seconds, followed by a final extension step of 72°C for 5 min. PCR products were visualised 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 and Qubit® double stranded DNA High Sensitivity Assay Kit (Invitrogen). RNA-seq library quality was assessed using an Agilent Bioanalyzer and Agilent High sensitivity DNA chip (Agilent) and confirmed that library insert sizes were ~200-250 bp for all individual libraries. Individual RNA-seq libraries were standardised 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.