Day 15 NI
RNA-Seq of TNG-MKOS-MEFs: non-infected, Day 15
Poly(A)+RNA was isolated directly from cell lysate with Dynabeads mRNA DIRECT Kit (61011, Life technologies), and sequencing libraries were prepared with the directional mRNA-seq sample preparation protocol from Illumina, with minor modifications. In brief, 50ng of poly(A)+ RNA was chemically fragmented with twice the volume of 50mM sodium carbonate [NaHCO3/Na2CO3], pH 9.2, 1mM EDTA at 95C for 5min, purified with RNeasy MinElute Cleanup Kit (74204, Qiagen), treated with thermosensitive alkaline phosphatase FastAP (EF0651, Fermentas) at 37C for 30min and then at 75C for 5min, incubated with ATP and T4 polynucleotide kinase (EK0032, Fermentas) at 37C for 1h and subsequently purified as before. RNA 3 adapter (RA3, part # 15013207, Illumina) and RNA 5' adapter (RA5, part #15013205, Illumina) were ligated as desribed in the Illumina protocol, each followed by RNA purification as described above. cDNA was synthesized using RNA RT Primer (RTP, part #15013981, Illumina) and SuperScript III (18080044, Invitrogen) as per Illumina protocol. Libraries were amplified for 14 cycles of PCR using forward (RNA PCR Primer (RP1), part #15005505 Illumina), and reverse (Illumina PCR Primer, Index) PCR primers. Reverse PCR primers with different indexes were used to allow multiplexing of libraries. Libraries were sequenced for 136 cycles on an Illumina HiSeq 2500 instrument.
Day 15 NI
SUB4139073
Day 15 NI