GSM5738120: siControl_MSU_rep3; Mus musculus; RNA-Seq
BMDM or mDM were lysed in TRIzol (ThermoFisher, #15596026). RNA isolation and DNase treatment was carried out using Direct-zol RNA MicroPrep kit (Zymoresearch, #11-33MB) 500ng-1 μg total RNA was enriched in poly-A tailed RNA transcripts by double incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and fragmented for 9 min at 94ºC in 2X Superscript III first-strand buffer containing 10mM DTT (Invitrogen, #P2325). The 10 μl of fragmented RNA was added to 0.5 μl of Random primers (Invitrogen, #48190011), 0.5 μl of Oligo d(T) primer (Invitrogen, #18418020), 0.5 μl of SUPERase inhibitor (Ambion, #AM2696), 1 μl of 10mM dNTPs and incubated at 50ºC for three minutes. Then, 5.8 μl of water, 1 μl of 10mM DTT, 0.1 μl of 2 μg/μl Actinomycin D (Sigma, #A1410), 0.2 μl of 1% Tween-20 (Sigma) and 0.5 μl of SuperScript III (Invitrogen, #ThermoFisher 18080044) was added to the mix. Reverse-transcription (RT) reaction was performed at 25ºC for 10 min followed by 50ºC for 50 min. RT product was purified with RNAClean XP (Beckman Coulter, #A63987) and eluted in 10 μl in 0.01% Tween-20. The RNA-cDNA complex was then added to 1.5 μl of 10X Blue Buffer (Enzymatics, #B0110-L), 1.1 μl of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 μl of 5U/μl RNAseH (Enzymatics, #Y9220L), 1 μl of 10U/μl DNA polymerase I (Enzymatics, #P7050L), 0.15 μl of 1% Tween-20 and 1.05 μl of nuclease free water; and incubated at 16ºC for 2.5h or overnight. The resulting dsDNA product was purified using 28 μl of SpeedbBead Magnetic Carboxylate (GE Healthcare, #651521050 50250) diluted in 20%PEG8000:2.5M NaCl to a final 13% PEG concentration, washed twice with 80% etOH, air dry and eluted in 40 μl of 0.05% Tween-20. The purified 40 μl of dsDNA was end-repaired by blunting followed by A-tailing and adapter ligation as described elsewhere (69) using BIOO Barcodes (BIOO Scientific, #514104), IDT TruSeq Unique Dual Indexes or Kapa Unique Dual-Indexed Adapters using 15 μl Rapid Ligation Buffer (Enzymatics, #L603-LC-L), 0.33 μl 1% Tween-20 and 0.5 μl T4 DNA ligase HC (Enzymatics, #L6030-HC-L). Libraries were amplified by PCR for 11-15 cycles using Solexa IGA and Solexa IGB primers (AATGATACGGCGACCACCGA and CAAGCAGAAGACGGCATACGA, respectively), purified using 1 μl of SpeedbBead Magnetic Carboxylate in 15.2 μl of 20%PEG8000:2.5M NaCl, washed with 80% etOH and eluted in 0.05% Tween-20. Eluted libraries were quantified using a Qubit dsDNA HS Assay Kit and sequenced on a NextSeq 500 or Hi-Seq 4000 (Illumina, San Diego, California). 