GSM2055130: S2_HSF_RNAi_NHS_PRO_seq; Drosophila melanogaster; OTHER
Nuclei isolation for PRO-seq: Nuclei isolation was performed using standard protocols. Briefly, cells were spun down, washed in cold 1x PBS once and then resuspended in 1mL of cold Swelling buffer (10mM Tris-Cl pH 7.5, 10% glycerol, 3mM CaCl2, 2mM MgAc2, 0.5mM DTT). After incubation on ice for 10 minutes, cells were transferred to a dounce homogenizer and 9mL of cold Lysis buffer (10mM Tris-Cl pH 7.5, 300mM sucrose, 10mM NaCl, 3mM CaCl2, 2mM MgAc2, 0.1% TritonX-100, 0.5mM DTT) was added. After douncing, the cells were spun down, washed in 10mL of cold Lysis buffer and resuspended in 1mL of cold buffer D (50mM Tris-Cl pH 8.0, 25% glycerol, 5mM MgAc2, 0.1mM EDTA, 5mM DTT). Next, cells were spun down again, resuspended in buffer D and flash frozen in liquid nitrogen.  RNA extraction for RNA-seq: Cells were spun down, washed in cold 1x PBS once and total RNA was extracted using TRIzol reagent and then isolated from the aqueous phase using the E.Z.N.A. Total RNA Kit I (Omega Bio-tek). PRO-seq library construction: PRO-seq libraries were prepared as described previously (Kwak et al., 2013). Briefly, 2×107 nuclei were added to 2x Nuclear Run-On reaction mix (10mM Tris-Cl pH 8.0, 5mM MgCl2, 300mM KCl, 1% Sarkosyl, 1mM DTT, 0.05mM each of biotin-11-A/C/G/UTP (PerkinElmer), 0.4 u/µL RNase inhibitor) and incubated for 3 min at 30°C. Total RNA was extracted and fragmented by base hydrolysis in 0.2N NaOH on ice for 10 min, and neutralized by adding 1x volume of 1M Tris-Cl pH 6.8. Fragmented nascent RNA was purified using streptavidin beads, ligated with reverse 3' RNA adapter (5'p-GAUCGUCGGACUGUAGAACUCUGAAC-/3'InvdT/), and biotin-labeled products were enriched by another round of streptavidin bead binding and extraction. For 5' end repair, the RNA products were successively treated with tobacco acid pyrophosphatase (TAP, Epicentre) and polynucleotide kinase (PNK, New England Biolabs). 5' repaired RNA was ligated to reverse 5' RNA adapter (5'-CCUUGGCACCCGAGAAUUCCA-3') before being further purified by a third round of streptavidin bead binding and extraction. RNA was reverse transcribed using the Illumina RP1 primer. TruSeq-barcoded libraries were generated by PCR amplification and products greater than 120 bp were PAGE purified and pooled for sequencing. RNA-seq library construction: PolyA+ RNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). TruSeq-barcoded RNA-seq libraries were generated with the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). Each library was quantified with a Qubit 2.0 (dsDNA HS kit; Thermo Fisher Scientific) and the size distribution was determined with a Fragment Analyzer (Advanced Analytical) prior to pooling.
GEO Accession
GSM2055130