Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: One ml of each sample stored in RNAlater, with OD above 0.6, was used in the procedure. Samples were centrifuged at 10,000 x g for 2 min on a bench centrifuge to collect the cell pellet. Pellet was washed twice with PBS buffer to remove RNAlater, and centrifuged at 10,000 x g. Pellets were later resuspended in 250 μl lysozyme (50 mg/ml final; Sigma) and 125 μl mutanolysin (1000 units/ml; Sigma), previously prepared and filter-sterilized in TE buffer (10 mM Tris, 1mM EDTA, pH 7.2). Samples were incubated at 37˚C for 10 min. Pre-lysed cells were centrifuged at 8000 x g for 1 min, and the Ambion RNAqeous kit (LifeTechnologies) was used to extract total RNA following manufacturer instructions and eluting the RNA in 50 μl of EB buffer. Total RNA was immediately subjected to DNase treatment with the Turbo DNase free (Ambion) using 3 μl of DNase I for 1 hour. RNA was stored at -80C at this point, but previously quantified in Qubit using the Qubit High Sensitivity RNA Assay Kit (Life Technologies). One μl of RNA was the minimum used in next steps. RNA integrity was checked in a Bioanalyzer using an Agilent RNA Nano Chip. RNA (1-2 µl) was first denatured at 65-70 ºC for 2 min before the run. Samples were further processed only if their RIN (RNA Integrity Number), was above 7.Ribosomal RNA depletion was achieved using the Ribo-Zero magnetic kit, Bacteria (Epicentre, Madison WA). Manufacturer instructions were followed (using 28 or 26 μl of RNA depending on the concentration). mRNA was purified with the Qiagen RNeasy Minelute Cleanup kit, following the instructions presented in the Ribo-Zero protocol for this part. RNA was eluted in 13 µl. mRNA was later converted to cDNA. For first strand cDNA conversion, the Superscript II Reverse Transcriptase was used (Invitrogen), following manufacturer protocol. Reaction also contained Random Primers (3 μg/μl, Invitrogen). We used 11 μl of rRNA depleted RNA, 1 μl of 10 mM dNTPs and 1 μl of random primers. After this, second cDNA strand was synthesized using the NEB NEBNext mRNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA) following manufacturer instructions. Then, the MinElute Reaction Cleanup Kit (Qiagen, Valencia CA) was used for cleaning up the DNA. Final elution was done in two rounds of 15 μl in EB buffer. At this point cDNA was quantified using the Qubit High Sensitivity DNA kit (Life Technologies), and diluted to a volume of 100 μl with EB buffer, for fragmentation in a Bioruptor (Diagenode). Eight cycles of 15 s on and 90 s off were used for each sample. Fragmented cDNA was quantified with Qubit High Sensitivity DNA kit. DNA was also analyzed in a Bioanalyzer with the Agilent High Sensitivity DNA kit. An average fragment length of 300-500 bp was desired for next steps. Sequencing libraries were prepared using the BiooScientific NEXTflex Chip Seq Kit (Bioo Scientific, Austin TX). This kit is Illumina compatible. Ten ng of fragmented cDNA were used. In the manufacturer protocol, option 3 was followed entirely, with the goal to select fragments between 300 and 400 bp in a gel-free protocol. Size selection and clean-up steps were achieved using the Agencourt Ampure XP beads (Beckman Coulter, Brea CA), and a Magnetic Stand-96 (LifeTechnologies). All temperature-sensitive reactions were carried out in a preheated thermocycler. Adapters used (NEXTflex™ ChIP-Seq Barcodes, Bioo Scientific) contained 6-bp indexes for multiplexing. At the last step, ligation products were amplified by PCR with the supplied reagents for 17 cycles. Pooled libraries were sequenced on an Illumina HiSeq 2500. Sequencing was run for 50 cycles, with read length of 50 bp (single reads).