title: GSM9293917: MALAT1 reporter pDual-GFP-(1-739) IRES-RFP, Modified, Replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: For RNA extraction, 900 µL TRIzol was added into each tube. Next, 180 uL of chloroform:isoamyl alcohol (24:1) was added and incubated for 5 min and subsequently centrifuged at 3,000 g for 15 min at 4°C. The aqueous phase was then transferred to a new tube and precipitated by EtOH (at a final concentration of 70%) overnight at -20°C. The following day, RNA was pelleted at 15,000 g for 30 min at 4°C and resuspended in 1x ME buffer (10mM MOPS, 0,1mM EDTA, pH 6.5) with 1:100 RNase inhibitor. RNA was then purified with the QIAGEN RNeasy Kit (Cat. No. 74104) and eluted with 1x ME with 1:100 RNase inhibitor. RT reactions were prepared by mixing 1 μg of total cellular RNA with 1uL of 1uM RT primer for each amplicon, annealed at 68°C for 5 min and cooled to room temperature. Next, 8uL of 2.5 x MarathonRT buffer (125mM 1M Tris-HCl pH 8.3, 500mM KCl, 12.5mM DTT, 1.25mM dNTPs, 2.5mM Mn2+), 4µL 100% glycerol and 0.5µL MarathonRT were added and incubated at 42°C for 3 h. The enzyme was then inactivated at 70°C for 15 min, followed by cDNA purification using AmpureXP at a 1.8x bead-to-sample ratio. Purified cDNA was eluted in 10uL nuclease-free water. Malat1 amplicons were generated using 5 μL of cDNA and gene-specific forward and reverse primers with NEB Q5 High-Fidelity 2x Master Mix under touchdown cycling PCR conditions (68 to 58 °C annealing temperature gradient). PCR products were purified with AmpureXP at a 1.8x bead-to-sample ratio and analyzed on a 1% agarose gel to verify the amplicon sizes. For library preparation, amplicon concentrations were measured with the Qubit dsDNA HS Assay Kit and amplicons diluted to 0.2 ng/μL. Libraries were generated using the Illumina NexteraXT DNA Library Preparation Kit according to the manufacturer's protocols, but at 1/5th the recommended volume. Library concentration and average fragment size were quantified with a Qubit dsDNA HS Assay Kitand a BioAnalyzer High Sensitivity DNA Analysis kit, respectively. Final libraries were diluted according to the manufacturer's protocols and sequenced on an Illumina NextSeq 2000 platform using a 300 cycle output kit.