title: GSM2828224: IP2_U2AF35; Homo sapiens; OTHER
library_construction_protocol: Exon-RBP complexes were assembled in a splicing competent nuclear extract prepared from HeLa cells; in transient transfection HeLa cells differentiated a sample of cloned mutant minigenes in a manner similar to HEK293 cells (Supplemental Fig_S20.jpg).  The saturation mutagenesis minigene library was PCR amplified with Phusion High-Fidelity DNA Polymerase (New England Biolabs) for a limited number of cycles (15) using the primers T7LibFwd TAATACGACTCACTATAGGACCTCTTCTTCTTTTCTAGA and LibRev gccagctagcACTCACTTG. The resulting T7 PCR DNA library was transcribed in vitro with MegaScript (Ambion) according to the manufacturer's protocol. The resulting saturation mutagenesis RNA library was cleaned with Quick Spin Sephadex Columns for RNA (Roche), purified by phenol/chloroform extraction and precipitated with ammonium acetate. Nuclear extract preparation was adapted from Hartmuth et al. . Briefly, HeLa cells were grown in suspension to 5 to 7 x105 cells per ml. Pelleted cells were Dounce homogenized in buffer A (10mM Hepes KOH pH7.9, 10mM KCl, 1.5mM MgCl2, 0.5mM DTT, 0.25mM PMSF). Resulting pelleted nuclei were dispersed in a Dounce homogenizer with 15 strokes of the B-type pestle in buffer C (20mM Hepes KOH ph7.9, 600mM KCl, 1.5mM MgCl2, 0.2mM EDTA pH8, 25% glycerol, 0.5mM DTT, 0.25mM PMSF). The nuclear extract obtained after centrifugation at 30,000xg for 40 min was dialyzed 2 x 2h against a minimum 50-fold volume of buffer D (20mM Hepes KOH pH 7.9, 0.1 M KCl, 0.2mM EDTA pH 8.0, 10% (v/v) glycerol, 0.5mM DTT, 0.25mM PMSF) , flash frozen in liquid nitrogen, stored at -80ºC and tested for splicing before performing RNA immunoprecipitation (IP). The microarray primer extension method and the subsequent double-stranded DNA linker ligation were described by Ray et al. (2009). A custom 60 bp 176,000 (4X44K) element microarray was purchased from Agilent. It was comprised of 5550 equally represented mutant sequences (555 per Hexmut) and 10 relative wild type sequences equally overrepresented at ~30 times the number of a mutant sequence. The 3' end of each 60‑mer was constant, representing 13 positions from -1 to +12 relative to the 3' end of the WT-1 exon 5 (Ke et al, 2011) with the sequence of “cttcttttctagA”. The remaining 47 nucleotides were specified as wild types or mutants, as described in Fig. 1 of the Results. The 19 nt double-stranded DNA linker with the sequence of “CAAgtgagtggacaatgcg” was ligated to the free end of the 60 nt double-stranded DNA probes. The eluted 79nt single-stranded DNA library (e.g. cttcttttctagAGTTGCTGCTGGGAGCTCCAGCACAGTGAAATGGACAGAAGGGCAGAGCAAgtgagtggacaatgcg) was then PCR amplified using the flanking primer pair F: ccccacctcttcttcttttctagA; R: the reverse complement of CAAgtgagtggacaatgcg), generating the 90nt double-stranded DNA library that was used to construct the 3kb full mini-gene library by overlap extension PCR (Fig 18). The relative wild type 60‑mer probe sequence of HMA is shown on line 1 below. All possible single and tandem double base changes were designed as shown in Fig. 1 and below: Line 1 shows the wild type sequence of HMA where the exon is underlined and the variable 6‑mer than distinguishes Hexmuts is double underlined. Starting at positon 2 of the exon, all 12 dinucleotides that changed the first base of the wild type dimer sequence were created (bold, lines 2 to 13). There are 15 possible mutant dinucleotides that can start at position 2; the 3 dimers missing in lines 2 to 13 appear when position 3 dimers are created, as seen underlined on lines 17, 21 and 25. The complete set of mutations covering the region 2 to 48 comprise 555 mutants, with the last position, 48, being necessarily comprised of only 3 single base substitutions. Incubations of the microarray were carried out in an Illumina hybridization oven with a rotating slide carrier. A 5'‑Cy3-labeled primer complementary to the 13 nt at 3' end of all microarray bound probes was extended with T4 DNA polymerase. The Cy3 label afforded confirmation of the double strandedness by examining the slide in a microarray scanner. The double stranded probes were then ligated  to a double stranded oligomer representing the 20 nt upstream of the 5' end of the sense strand of the probe. The sense strand of the 20‑mer carried a Cy5 label at the 5' end and the antisense strand carried a ddC appended to the 3' end to prevent primer self ligation and a phosphate included on the 5' end for ligation to the probe. The Cy5 label afforded confirmation of the ligation efficiency by scanning. The single stranded extension-ligation products were stripped from the slide with NaOH  at 65C for 20 minutes and used as a mega-primer for minigene construction by overlap extension PCR as shown in Supplemental_Fig_S18.jpg.
identifiers.namespace: GEO
identifiers.id: GSM2828224