title: LIN28 CSD RNA Bind and Seq after 1st round of selection
design: The LIN28A CSD (aa 1-120) with an N-terminal FLAG-HA-tag was transiently expressed in HEK293 cells for 48h. Cells from two 15cm culture dishes were harvested and lysed in 1 ml IP lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM KCl, 1 mM AEBFS, 1 mM DTT, 0.5% (v/v) NP-40). Insoluble material was pelleted by centrifugation (20,000g, 4C, 15min) and the supernatant transferred to a fresh reaction tube containing 20 l FLAG-M2 Agarose Beads (Sigma). The binding reaction was incubated at 4C for 2-3 hours while agitating. The beads were then washed three times with 1 ml IP wash buffer (50 mM Tris-HCl, pH 7.5, 300 mM KCl, 0.05 % (v/v) NP40). The immunoprecipitated proteins were used directly in an RNA-selection reaction by resuspending the beads in 400 l binding buffer (25 mM Tris Cl pH 7.5, 150 mM KCl, 3 mM MgCl2, 0.01% (v/v) NP 40, 1 mg/ml BSA, 1 mM DTT, 5% (v/v) glycerol, 0.1U/l Ribolock) and adding 10g of an RNA-pool of the sequence 5-NNNNNNNNGUUU-3. The binding reaction was incubated for 30 minutes at room temperature with agitation. Beads were then collected by centrifugation (1000 g, 2 min, 4C) and washed three times with ice-cold binding buffer. Bound RNA was eluted in 200 l elution buffer (10 mM Tris-Cl pH 7.0, 400 mM NaCl, 1 mM EDTA, 1% (w/v) SDS) and purified by phenol-chloroform extraction and ethanol precipitation.   The selected RNA molecules were sequentially ligated to a 3 DNA adaptor and a 5RNA adaptor containing a T7 promoter sequence. The ligated product was reverse transcribed using the First Strand cDNA Synthesis Kit (Thermo) and the primer 5 GCCTTGGCACCCGAGAATTCCAGTTT-3. A PCR reaction was used to amplify the cDNA sequence and introduce barcodes for next generation sequencing (NGS).  To separate insert containing amplification products from empty adaptor sequences, the PCR reaction was run on a 6% Urea PAGE gel and the band at 150bp corresponding to the desired product was excised. The DNA was eluted overnight in 0.4 M NaCl and precipitated with ethanol. The re-dissolved PCR product was stored for NGS analysis.   To generate RNA for a second round of selection, 50 ng of the PCR-pool was amplified using the primers 5 AATGATACGGCGACCACCGAGATCTACACGTTCAGTAATACGACTCACTATAGG-3 and 5-GCCTTGGCACCCGAGAATTCCAGTTT-3. The resulting PCR-product was purified using a PCR Clean-up Kit (Macherey Nagel) and cleaved by addition of 1.5 l Fast digest MssI (Thermo), which recognizes the restriction site GTTTAAAC that is generated by the ligation of the RNA insert with the 3adaptor. The cleaved DNA was transcribed with T7 polymerase, which yields a new pool of RNAs with the sequence GGGNNNNNNNNGUUU. The RNA was purified by 18% Urea PAGE, dephosphorylated with FastAP (Thermo) and monophosphorylated with polynucleotide kinase. 10 g of the prepared RNA were used in a second selection cycle with freshly immunoprecipitated LIN28A CSD, ligated and amplified as described above.  Libraries from once and twice selected RNA were sequenced on a MiSeq instrument (Illumina) with a 150 cycle MiSeq Reagent Kit to which we added a custom Read1 sequencing primer (5-GATCTACACGTTCAGTAATACGACTCACTATAGGG-3).
identifiers.namespace: SUB4119054
identifiers.id: LIN28_CSD_1st_round