GSM4816429: 24h Hippo-IR rep3; Drosophila melanogaster; RNA-Seq
For each replicate flight muscles from seven Mef2-GAL4, UAS-GMA-GFP pupae at 24 h and 32 h APF were dissected in ice-cold PBS treated with DEPC using a fluorescent binocular. Flight muscles were collected in an Eppendorf tube and centrifuged at 2000g for 5 min. The flight muscle pellet was re-suspended in TRIzol™, shock-frozen in liquid nitrogen and kept at -80°C. RNA was isolated directly from the TRIzol muscle samples using a 96 well plate extraction kit (Direct-zol™-96 RNA, Zymo Research, #R2054): after thawing to room temperature in 1,5ml Eppendorf tubes, the tissue samples were homogenized using a small pestle, followed by nucleic acid precipitation with 100% ethanol. The pellet was dissolved and transferred to the 96 well plate containing the purification columns. DNA digestion was performed in columns according to the kit instructions. Total RNA was eluted with 25 μl of RNase-free water and was quantified using the Quantifluor RNA System (Promega, #E3310). RNA sequencing libraries were prepared using 20 ng of total RNA following the BRB-sequencing protocol. Briefly, each RNA sample was reverse transcribed in a 96-well plate using SuperScriptTM II Reverse Transcriptase (Lifetech 18064014) with individual barcoded oligo-dT primers (Microsynth, Switzerland). Next, the samples were split into 3 pools, purified using the DNA Clean and Concentrator kit (Zymo Research #D4014), and treated with exonuclease I (New England BioLabs, NEB #M0293S). Double-stranded cDNA was generated by the second strand synthesis via the nick translation method. For that, a mix containing 2 μl of RNAse H (NEB, #M0297S), 1 μl of E. coli DNA ligase (NEB, #M0205 L), 5 μl of E. coli DNA Polymerase (NEB, #M0209 L), 1 μl of dNTP (0 .2 mM), 10 μl of 5x Second Strand Buffer (100 mM Tris, pH 6.9, AppliChem, #A3452); 25 mM MgCl2 (Sigma, #M2670); 450 mM KCl (AppliChem, #A2939); 0.8 mM β-NAD Sigma, N1511); 60 mM (NH4)2SO4 (Fisher Scientific Acros, #AC20587); and 11 μl of water was added to 20 μl of ExoI-treated first-strand reaction on ice. The reaction was incubated at 16 °C for 2.5 h. Full-length double-stranded cDNA was purified with 30 μl (0.6x) of AMPure XP magnetic beads (Beckman Coulter, #A63881) and eluted in 20 μl of water. The Illumina compatible libraries were prepared by tagmentation of 5 ng of full-length double-stranded cDNA with 1 µl of in-house produced Tn5 enzyme (11 μM). After tagmentation the libraries where purified with DNA Clean and Concentrator kit (Zymo Research #D4014) eluted in 20 µl of water and PCR amplified using 25 μl NEB Next High-Fidelity 2x PCR Master Mix (NEB, #M0541 L), 2.5 μl of P5_BRB primer (5 μM, Microsynth), and 2.5 μl of Illumina index adapter (Idx7N5 5 μM, IDT) following program: incubation 72 °C—3 min, denaturation 98 °C—30 s; 15 cycles: 98 °C—10 s, 63 °C—30 s, 72 °C—30 s; final elongation at 72 °C—5 min. The fragments ranging 200–1000 bp were size-selected using AMPure beads (Beckman Coulter, #A63881) (first round 0.5x beads, second 0.7x). The libraries were profiled with High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, #DNF-474) and measured with Qubit dsDNA HS Assay Kit (Invitrogen, #Q32851) prior to pooling and sequencing using the Illumina NextSeq 500 platform using a custom primer and the High Output v2 kit (75 cycles) (Illumina, #FC-404-2005). The library loading concentration was 2.2 pM and sequencing configuration as following: R1 6c / index 8c / R2 78c.
GEO Accession
GSM4816429