Micro-RNA sequencing of Ago-APP samples from neuroblasts, neurons or glial cells of L3 Drosophila melanogaster larvae CNS
Micro-RNA sequencing of Ago-APP samples from neuroblasts, neurons or glial cells of L3 Drosophila melanogaster larvae CNS
A transgenic Drosophila line expressing T6B-FHY under the control of UAS sequences was generated, allowing cell-type specific expression of the fusion protein when combined with suited GAL4 driver lines (Fig.1B)26. For temporal control, we combined this approach with the GAL80 temperature sensitive system (tubulin-GAL80ts). Thus, when x-GAL4, tub-GAL80ts, UAS-T6B animals are maintained at 18° (restrictive temperature), GAL4-dependent expression is prevented. Switching to 29° (permissive temperature) for 24 hours allows GAL4-dependent expression (Fig.1B,C).  For cell type specific expression, we combined this temporal control system with well characterized  driver lines for i. neuroblasts (nab-GAL4/tub-Gal80ts), ii. the entire neuronal lineage comprising  predominantly neurons (elav-GAL4/tub-Gal80ts) and iii. glial cells (repo-GAL4/tub-Gal80ts).  For AgoAPP isolation, 20 late larval CNS were dissected 24h after induction of T6B expression for each transgenic combination. Samples input1 to 3 were generated after gel fractionation based on size corresponding to micro-RNA length. Samples elav1 to 3, repo 1 to 3, nab 1 to 3 were generated by AgoAPP. T6B-FHY expressing Drosophila CNS were isolated and fixed with 4% PFA for 10 minutes and subsequently quenched in a final concentration of 150 mM Glycine for 5 minutes, at room temperature each. After washing twice with ice-cold PBS, CNS were shock-frozen and stored at -70 °C until use. Ago-APP was performed according to previously published protocol with minor modifications. Tissue was lysed in 1ml lysis buffer (150 mM KCl, 25 mM Tris, pH 7.5, 2 mM EDTA, 0.5% Nonidet P-40; supplemented with 1 mM NaF, 1 mM DTT and 1 mM AEBSF before use) and sonicated (Vibra cell (75042), Bioblock Scientific) for 8 cycles, 10 seconds on, 10 seconds off at an amplitude of 34%. Lysation was cleared by centrifugation (15'000 x g for 15 min at 4 °C). 50 ul of lysate was kept for input control. A maximum of 500 ug total protein in a volume of 950 ul was loaded per 25 ul GFP-Trap agarose beads (gta, Chromotek), prior washed with ice-cold PBS (2 min at 2'500 x g) and incubated for 1 hour rotating at 4 °C. IP was washed five times with ice-cold washing buffer (1 M NaCl, 50 mM Tris, pH 7.5, 5 mM MgCl2, 0.01% Nonidet P-40; supplemented with 1 mM NaF, 0.5 mM DTT and 0.5 mM AEBSF before use), transferred into a new tube and washed once with ice-cold PBS. For RNA analysis, the IPed complexes were released from the beads by incubating the pellet for 10 minutes with 50ul 4% SDS in 0,1M NaHCO3 (same treatment was performed with the input sample). Supernatant was collected and the beads-releasing process was repeated. To reverse crosslink the formaldehyde linking, the samples were treated in 20 mg/ml Proteinase K (Roche) in PK buffer (100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM EDTA) (pre-incubated for 20 minutes at 37 °C to eliminate potential RNases) at 65 °C overnight, shaking at 1000 RPM. RNA was isolated with Trizol (Invitrogen). miRNA Seq was performed according to the AQ-Seq with minor modifications. In brief, for small RNA library preparation 50 to 300 ng of total RNA was ligated with randomized 3' adapter using T4 RNA ligase 2 truncated KQ (NEB) in 1X T4 RNA ligase reaction buffer (NEB) supplemented with 20% PEG8000 (NEB). Ligated miRNA-adapter fragments were gel-purified and eluted in 0.3 mM NaCl, 2mM EDTA (pH8). After centrifugation the EtOH-precipitated fragments were resuspended in water and ligated to 0.25 µM randomized 5' RNA adapter using T4 RNA ligase 1 (NEB) in the same buffer conditions as described above. Reverse transcription with Superscript III First-Strand synthesis supermix (ThermoFisher Scientific) and 5 µM RT Primer (RTP, TruSeq kit; Illumina) was then performed according to the manufacturer's recommendations. After cDNA amplification the PCR amplicons representing miRNA sized inserts were gel-purified, precipitated and resuspended in water. The quality of the libraries was assessed by qPCR