title: GSM3073876: Srrm4 expression reporter, sorted cells with 2% lowest EGFP, replicate2 (T16); Mus musculus; OTHER
library_construction_protocol: cell engineering protocol: Srrm4 was endogenously EGFP-tagged at its N-terminus in N2A cells using CRISPR-directed targeted genomic insertion, as described previously (Mali et al., 2013; Ran et al., 2013). In brief, N2A cells were co-transfected with pX459 plasmid expressing Cas9 and an sgRNA targeting Srrm4 near the translation initiation site, as well as with a repair template, using Lipofectamine 2000 (Invitrogen). The repair template containing the EGFP sequence, flanked by 800-900 bp homology arms corresponding to the up- and down-stream sequences of the Srrm4 translation initiation site, was cloned into a pBluescript vector (with the hPGK driven Blasticidin resistance gene inserted). The homology arms were flanked with DNA sequences corresponding to the sgRNA target sequence so that the vector would be linearized in cells. The PAM sequence of the sgRNA sequence upstream of the ATG was mutated in the repair template so as not to be targeted by Cas9. For generating the knock-in lines the following sgRNA target sequence was used: 5´-GTTTCACGCGGACAGCGCCC(CGG)-3´.  Cells were selected for 3 days with Puromycin (2.5 μg/ml) and Blasticidin S (10 μg/ml) for pX459 and pBluescript transfection, respectively, and cultured for seven more days before single cell sorting for EGFP+ cells with a BD Influx FACS sorter (BD Biosciences). The insertion sequence in each sorted clone was confirmed at the genomic level using Sanger sequencing, and clones were tested for EGFP-Srrm4 expression using fluorescent microscopy, western blotting, and 3´ RACE PCR (Scotto–Lavino et al., 2007). 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: GSM3073876