Illumina HiSeq 2500 sequencing; 5'-end mapping of total Mycoplasma pneumoniae RNA
5'-end mapping of total Mycoplasma pneumoniae RNA
Mycoplasma pneumoniae M129 cells were grown to exponential phase in modified Hayflick medium, washed with PBS and collected in lysis buffer. miRNeasy kit from Qiagen (with in column DNase digestion) In order to obtain transcriptome-wide, the mRNA 5' ends (TSSs), we amplified cDNA using a random primer by RT-PCR. Specifically RT_read2 at 2.5 µM was hybridized to 5 µg of total RNA by heating to 65 ºC for 5 min and quick chilling on ice in a 11 µl total volume. Retrotranscription was performed by adding 4 μl 5X first-strand buffer, 2 µl 0.1 M DTT, 1 µl RNase OUT (40 units/µl, Promega), 1 µl 10 mM dNTP mix, and 1 µl SuperScript II RT (200 units, Invitrogen) and incubating for 50 min at 42C before inactivation at 70C for 15 min. Once the reaction was finished, RNA was removed by first adding 1 µl of 4 N NaOH during 5 min at 95ºC, then neutralizing it with 2 µl of 1 M Tris•HCl (pH 8). Then we added 1 µl of RNase cocktail (Ambion) and incubated the tube at 37ºC during 30 min. After this, the reaction was brought to 50 µl with H2O and the ssDNA was cleaned with 1 volume of AMPure beads as above. Finally, we eluted with 25 µl of EB and proceeded to the ssDNA ligation. We prepared a 50 µl reaction with 5 µl of 10x buffer, 1 µl of ATP, 2.5 µl of MnCl2, 2 µl of CircLigase (Epicenter), 0.84 µl of 100 µM linker (L_read1_N6) and 15 µl ssDNA (from previously step). This mix was incubated at 65ºC during 120 min, and then the ligase inactivated at 85ºC during 15 min. The reaction was cleaned with 1 volume AMPure beads as above and eluted with 25 µl of EB. The final step involved a PCR reaction in order to introduce Illumina sequences and thus prepare the libraries for sequencing. This reaction was performed with 4 µl of 5x Buffer, 0.4 µl of dNTPs, 0.4 µl of Phusion HF polymerase (NEB), 0.5 µM of primers (F_PEu and indexed R_PE) and 5 µl of ssDNA in a 20 µl final reaction volume. We PCR-amplified during 20 cycles (elongation at 60ºC). Each PCR product was checked on a 2% agarose/TAE gel, and once the correct band was obtained (aprox. 250 bp), we repeated the same reaction but in a 50 µl final volume. The PCR was cleaned and selected by size using AMPure beads (as above), and quantified by qPCR for the ultrasequencing process. Oligos:  RT_read2 CAGACGTGTGCTCTTCCGATCNNNNNN L_read1 [Phos]NNNAGATCGGAAGAGCGTCGTGTAG[SpcC3] F_PEu AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T R_PEi6 CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC R_PEi12 CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC