Illumina HiSeq 2000 sequencing; Identification of RNA targets of EB1 using RIP-seq in Drosophila ovaries
Identification of RNA targets of EB1 using RIP-seq in Drosophila ovaries
Ovaries from EB1-GFP and GFP alone expressing flies were harvested in PBS and resuspended in lysis buffer (10mM TrisCl pH7.5, 150mM NaCl, 0.5mM EDTA, 0.5% NP40, 0.5% SDS, freshly added protease inhibitor and ribolock). They were crosslinked with UV (0.3 J/sq cm), followed by homogenisation and lysis. The samples were centrifuged at max speed for 10 minutes and the supernatant was incubated with GFP-Trap MA beads for 2.5 h, followed by washing with high salt buffer (10mM TrisCl pH7.5, 750mM NaCl, 0.5mM EDTA, 0.5% NP40, 0.5% SDS, 0.02mg/mL heparin, freshly added protease inhibitor and ribolock), medium salt buffer (10mM TrisCl pH7.5, 500mM NaCl, 0.5mM EDTA, 0.5% NP40, 0.5% SDS, 0.02mg/mL heparin, freshly added protease inhibitor and ribolock) and finally low salt buffer (10mM TrisCl pH7.5, 750mM NaCl, 0.5mM EDTA, 0.5% NP40, 0.5% SDS, 0.02mg/mL heparin, freshly added protease inhibitor and ribolock). This was followed by proteinase K treatment and RNA extraction using Trizol, following manufacturer's instructions. Quality of RNA samples was checked with Bioanalyzer Pico chip assay. cDNA was prepared from 1ng of total RNA using the Smart-Seq 2 method with 20 pre-amplification PCR cycles, as described by Picelli et al. (2014)(1). NGS libraries were prepared using the home-made tagmentation-based method as described by Hennig et al., (2018)(2). Briefly, 125 ng of cDNA was tagmented using home-made Tn5 loaded with annealed linker oligonucleotides for 3 minutes at 55C. Reaction was inactivated by adding 1.25microl of 0.2% SDS and incubation for 5 minutes at room temperature. Indexing and amplification was done using the KAPA HiFi HotStart PCR kit (Sigma-Aldrich) with Index oligonucleotides (sequences were adapted from Illumina).    (1)Simone Picelli, Omid R Faridani, ??sa K Bj??rklund, G??sta Winberg, Sven Sagasser & Rickard Sandberg. 2014. Full-length RNA-seq from single cells using Smart-seq2. Nature Protocols. 9: 171???181.??    (2)Bianca P. Hennig, Lars Velten, Ines Racke, Chelsea Szu Tu, Matthias Thoms, Vladimir Rybin, H??seyin Besir, Kim Remans and Lars M. Steinmetz. 2018. Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3: Genes, Genomes, Genetics. 8 (1): 79-89.