GSM4396365: Balb/c_WT_Cocci_infected_1; Mus musculus; RNA-Seq
At 14 days post-infection, lungs were rapidly removed and one lobe of the left lung was immediately frozen in liquid nitrogen and stored at −70°C. Lung tissue was thawed in the presence of Trizol (Life Technologies) and homogenized using the Tissuelyser II (Qiagen). RNA was isolated with Trizol according to the manufacturer's instructions, DNase-treated using TURBO DNase (Ambion) according to the manufacturer's instructions and ethanol-precipitated. PolyA-RNA was selected from total RNA using the MicroPoly(A)Purist kit (Ambion), according to the manufacturer's instructions. Isolated RNA was hydrolyzed in a total volume of 20 μl with 2 μl RNA fragmentation buffer (Ambion) for 10 min at 70 °C. The reaction was stopped with stop buffer, and buffer was exchanged to Tris, pH 8.5, using P30 size-exclusion columns (Bio-Rad). The fragmented RNA (30 ng) was 5′-decapped in a total volume of 21 μl containing 0.5 μl tobacco acid pyrophosphatase (TAP, Epicentre), 2 μl 10× TAP buffer and 1 μl SUPERase-IN, and incubated for 2 h at 37 °C. To dephosphorylate RNA 3′ ends, 0.5 μl 10× TAP buffer, 1.5 μl water, 0.5 μl of 0.25 M MgCl2 (4.17 mM final; 1 mM EDTA for maximum phosphatase activity), and 0.5 μl of 10 mM ATP (0.2 μM final to protect PNK) where added, and the reaction was incubated with 1 μl PNK (Enzymatics) for 50 min at 37 °C. RNA fragments were 5′-phosphorylated by adding 10 μl 10× T4 DNA ligase buffer, 63 μl water and 2 μl PNK, and incubated for 60 min at 37 °C. RNA fragments were isolated using Trizol LS, precipitated in the presence of 300 mM sodium acetate and 2 μl glycoblue (Ambion), washed twice with 80% ethanol and dissolved in 4.5 μl water. To prepare sequencing libraries, 0.5 μl of 9 μM 5′-adenylated 3′MPX adaptor/5Phos/AGATCGGAAGAGCACACGTCTGA/3AmMO/ (IDT, desalted; adenylated with Mth ligase (NEB) according to the manufacturer's instructions, phenol–chloroform/chloroform-extracted, ethanol-precipitated with glycogen and dissolved in water at 9 µM) was heat-denatured together with the RNA for 2 min at 70 °C, and ligated with 100 U truncated T4 RNA ligase 2 K227Q (NEB) in 10 μl 1× T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 h at 16 °C. To reduce adaptor dimer formation, 0.5 μl of 10 μM MPX_RT primer 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ (IDT, desalted) was added and annealed to the ligation product by incubating at 75 °C for 2 min, then 37 °C for 30 min, and 25 °C for 15 min. Finally, 0.5 μl of 5 μM hybrid DNA/RNA sRNA 5′h adaptor 5′-GTTCAGAGTTCTACArGrUrCrCrGrArCrGrArUrC-3′ (IDT) was ligated to previously capped RNA 5′ ends by adding 2 μl T4 RNA ligase buffer, 6 μl 50% PEG8000 (15% final), 1 μl of 10 mM ATP, 9.5 μl water and 0.5 μl (5 U) T4 RNA ligase 1 for 90 min at 20 °C. To 15 μl of ligation reaction, an additional 0.5 μl of 10 μM MPX_RT primer was added, reactions were denatured at 70 °C for 1 min and then placed on ice. RNA was reverse-transcribed by adding 3 μl 10× first-strand buffer (4.5 µl water, 1.5 μl of 10 mM dNTP, 3 μl of 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen)), and incubating for 30 min at 50 °C. Complementary DNA was isolated by adding 35 μl AMPure XL beads (Beckman), binding and washing according to manufacturer's instructions, and dissolving in 40 μl TET (0.1% Tween 20 in TE buffer). Libraries were PCR-amplified for 12-15 cycles, with 0.75 μM oNTI201 primer and TruSeq-compatible indexed primers using Phusion Hot Start II in Phusion HF buffer (Thermo Scientific) containing 0.5 M betaine (98 °C, 30 s; 12× (98 °C, 10 s; 57 °C, 25 s; 72 °C, 20 s); 72 °C, 1 min; stored at 4 °C, and 225–400-bp fragments were size-selected on 10% PAGE gels. Libraries were diluted with TET buffer and quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), pooled and sequenced on a Hi-Seq 2000 sequencer (Illumina) using single end 50bp reads.
GEO
GSM4396365