title: GSM3073863: Mef2d splicing reporter, sorted cells with 30% lowest EGFP/mCherry, replicate 1 (T9); Mus musculus; OTHER
library_construction_protocol: cell engineering protocol: A Flp-in cassetted was integrated into the Rosa26 locus in N2a cells by first creating cells with an FRT site in that locus, followed by integration of a Flp-In construct from pcDNA5/FRT/TRE modified to include an rtTA3-compatible Tet Response Element (TRE). A bichromatic microexon splicing reporter based on a microexon from either murine Shank2 (ENSMUSE00001006087) or Mef2d (ENSMUSE00000673645) was then constructed by amplifying the microexons (extended by one nucleotide to cause a frame shift), flanking introns, upstream constitutive exon, and 20-50 nt of the downstream constitutive exon 5' of a fluorescence cassette consisting of EGFP and mCherry in different reading frames. This construct was cloned into a customized pcDNA5-based Gateway(R) compatible vector (Life Technologies) that contained 8x TET response elements followed by a miniCMV promoter, Kozak sequence, 3x Flag-tag, and attR sites, followed by integration in a clonal N2A Flp-In cell line. Genomic DNA was extracted from the cell pellets of unsorted samples using the QIAamp Blood Maxi Kit (Qiagen) while gDNA from sorted populations was purified with the Midi Kit as per the manufacturer's recommendations. Genomic DNA was precipitated using ethanol and sodium chloride, and resuspended in Buffer EB (10 mM Tris-HCl, pH 7.5). gRNA inserts were amplified via PCR using primers harboring Illumina TruSeq adapters with i5 and i7 barcodes, following a two-step PCR approach.   In the first step, a total of 50 μg of gDNA was subjected to PCR (25 cycles, temperature of annealing (Ta) = 65°C) for enrichment of sgRNA cassettes using NEBNext Ultra II Q5 polymerase and staggered primers annealing to the end of the U6 promoter and the beginning of the tracrRNA. For the 2% highest or lowest expressing sorted cell populations 10 μg of gDNA was used. Subsequently, the individual PCR reactions were pooled and 50 μl of the first step PCR product was loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1.5 hour at 100V. PCR products of 200-230 bp were selected and the excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). 1/6th of the purified PCR products were subjected to 2nd step PCR (10 cycles, Ta= 62°C) using NEBNext Ultra II Q5 polymerase and primers harboring Illumina TruSeq adapters with i5 and i7 indices to generate barcoded sequencing libraries ready for Illumina sequencing (Table S8). The PCR amplicons were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100V. The libraries were size-selected at 270 bp and 300 bp, and the excised gel fragments were purified using Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen).  The purified libraries were analyzed for quality control, pooling and sequencing. Size confirmation analysis for each sample was performed on an Agilent Bioanalyzer dsDNA High Sensitivity chip. Amplicons were quantified using Kapa Universal qPCR Master Mix (Cat # KK4923, Roche) and Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and pooled at varied ratios by molarity after size-adjustment. To mitigate the effects of index hopping, pooling was performed just prior to sequencing. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (Cat # E7630L, NEB).
attributes.tag: GEO Accession
attributes.value: GSM3073863