title: Cell-type and subcellular compartment specific isolation of miRNAs directly from the mouse brain by in vivo AGO-APP
design: Cell-type and subcellular compartment specific isolation of miRNAs directly from the mouse brain by in vivo AGO-APP
library_construction_protocol: Samples were issued from brain tissues of transgenic mice expressing a T6B peptide either in the whole cytoplasm (T6B-FHY) or targeted to the post-synapse (PSD95_T6B-HFY). These 2 transgenic mice were bred with the Nestin-CreERT2 transgenic mouse line to extract miRNAs from the olfactory bulb interneurons and with the NeuroD6-CreERT2 transgenic mouse line to extract miRNAs from the excitatory neurons of the cortex. From the 4 combinations of transgenes we extracted the micro-RNAs bound to argonaute using the Ago-APP protocol.  Olfactory bulb or cortex tissues were dissected from T6B or PSD95_T6B expressing mouse brains and immediately fixed in 1 ml 4% paraformaldehyde (PFA) for 10 minutes at 25 °C. Fixation was quenched in 245 mM glycine for 5 minutes at 25 °C and samples were washed twice with ice-cold phosphate-buffered saline (PBS) for 5 minutes at 25 °C.  Fixed tissue samples were then lysed in 1 ml of lysis buffer containing 150 mM KCl, 25 mM Tris-HCl (pH 7.5), 2 mM EDTA, supplemented freshly with 1 mM NaF, 0.5% NP-40, 1 mM DTT, and 1 mM AEBSF. Cell lysis was performed by sonication using a Vibra Cell sonicator (10 cycles of 1 minute and 20 seconds each; 10 seconds on, 10 seconds off; 34% amplitude; 4 °C). The lysate was clarified by centrifugation at 15,000 × g for 15 minutes at 4 °C.  Total protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Product Number: 23227), and the lysate was adjusted to a final concentration of 2000 µg in a total volume of 1000 µl.  For each immunoprecipitation (IP), 25 µl of GFP-TRAP® Agarose (GTA) beads were used. The beads were blocked with 1% BSA for 2 hours at 4°C, followed by two washes with ice-cold PBS, and centrifuged for 2 minutes at 2500 × g. A total of 950 µl (1900 µg) of cleared lysate was added to the washed beads, and the mixture was incubated for 1 hour at 4°C with shaking or on a rotator wheel.  The beads were then washed five times with wash buffer containing 1 M NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, supplemented with freshly added 1 mM NaF (Sigma-Aldrich, Product Number: PHR1408-1G), 0.01% NP-40, 0.1–1 mM DTT (Sigma-Aldrich, Product Number: 3483 12-3), and 0.1–1 mM AEBSF (Sigma-Aldrich, Product Number: SBR00015-1ML). During the final wash step, the beads were transferred to a new tube precoated with PBS for at least 1 hour to prevent bead sticking. After the final wash, the beads were washed once more with ice-cold 1X PBS. IP samples were supplemented with 50 µl of 4% SDS in 0.1 M NaHCO₃, and incubated at 50 °C for 10 minutes with shaking at 700 rpm. The supernatant was collected, and the bead release was repeated to ensure complete recovery. Proteinase K (PCR grade, Roche, Product Number: 03115801001) was prepared at 20 mg/ml in PK buffer and pre-incubated at 37 °C for 20 minutes to inactivate potential RNases. A total of 100 µl of the prepared Proteinase K solution was added to each sample (input and IP), and the mixture was incubated overnight at 65 °C with shaking at 1000 rpm. RNA was then extracted with 1 ml of TRIzol reagent following the manufacturer protocol. At the end of the process the RNA pellets were resuspended in 12 µl of nuclease-free water.  9 µl of the AGO-APP samples were first polyadenylated according to the manufacturer (NEB) instructions. Following enzyme deactivation (65°C for 20 minutes), RNA precipitation with ethanol and resuspension in 2.5 µl of water, a 5′ randomized adapter (GUUCAGAGUUCUACAGUCCGACGAUCNNNN) was ligated to the polyadenylated miRNAs using T4 RNA-Ligase 1 according to manufacturer (NEB) instructions in a final volume of 10 µl. For this ligation step the adapter concentration was adapted to the amount of miRNA in the sample as estimated by the qRT-PCR previously realized. Adapting the adapter concentration allows to favor the amplification of the miRNA containing molecules. Bellow, the rule we followed: CT of let-7a-5p  5' adapter concentration 14-17   0.18 µM 18-19   0.05 µM 19-20   0.01 µM 21-25   0.003 µM Subsequently 3µl of the ligation reaction was directly reverse-transcribed using the v-dT20-RA3 (GCCTTGGCACCCGAGAATTCCATTTTTTTTTTTTTTTTTTTTV ) oligonucleotide to prime the reaction. Then the library was amplified by PCR performed on 5 µl of the ligation reaction using the Illumina primers RP1 and an RPIx index primer and following the program: 98° for 1 min - X times (98°C for 10 seconds, 58°C for 30 seconds, 72°C for 15 seconds) - 72°C for 10 min. The number X of cycles was previously determined through pilot PCR reactions performed on 1 µl of the reverse transcription reaction under the same amplification conditions but applying a variable number of cycles: 18, 21 and 25. The products of the PCR reaction were loaded on a 6% polyacrylamide TBE gel, from which the desired band of approximately 165 bp was extracted and purified. After purification, the concentration of all samples of the library was analyzed on an Agilent 2100 Bioanalyzer, according to the manufacturer's instruction