GSM5682066: HDR Pool 4 GL2 - Rep 2 gfp -; Homo sapiens; OTHER
DNeasy Blood & Tissue Kit, eluted in 200 ul H2O. To amplify the barcode from gDNA from the GFP-positive and negative populations were spread over 8-16 reactions containing 250 ug of gDNA each (220K‚Äì440K genome equivalents by weight given HeLa triploid genome ~9 pg). Reactions also contained Kapa2G Robust Polymerase (Kapa Biosystems), a primer that annealed to the SV40 promoter 5‚Äô of the integrated plasmid and a primer adjacent to the barcode (SV40_F and newpc5bc_nexteraR). PCR was performed using the following conditions [95C, 5 min; {95C, 40s, 65C, 30s, 72C, 3 min} x ~28 cycles; 72C, 10 min]. The reactions produce a ~3,700 base amplicon specifically from integrated plasmids. The reactions for each sample were combined and the amplicons were purified by 0.5X Ampure and eluted with 10 mM Tris at 10% of the original reaction volume. 10% of the eluted PCR volume was re-amplified with primers containing sample indexes and Illumina cluster generating sequences (pc5bc_p5_F, nextIndex). Reactions also contained Kapa2G Robust Polymerase and 0.5X SYBR green II (ThermoFisher). PCR reactions are monitored on a Mini-opticon qPCR machine (Bio-Rad) and removed during exponential amplification using the following conditions [95C, 3 min; {95C, 20s, 65C, 30s, 72C, 20s} x 5-10 cycles]. The PCR reaction produced a 350-base amplicon that was purified using a double-Ampure protocol. First the large DNA fragments were removed by precipitation by addition of 0.6X volume Ampure beads to the reactions. Then the 350-base amplicons were purified from the supernatant using 0.9X volume Ampure beads. The samples were multiplexed and the barcodes and sample indexes were sequenced (single read, pcDNA5_barcodeSeq_F) on a Nextseq 500 High Output 75 base kit. pcDNA5-FRT/TO-HA-BRCA1 was digested with NheI-HF and SbfI-HF (New England Biosciences), and the 1700 bp fragment containing amino acids 1294-1863 was cloned into the pUC19 plasmid. For experiments mutagenizing amino acids 1557-1768, the pcDNA5-FRT/TO-HA-BRCA1 plasmid was modified to include regions of homology for Illumina sequencing (pcDNA5-FRT/TO-HA-BRCA1-Blue). Three site-saturation mutagenesis libraries – pool 4 (amino acids 1769-1863), pool 5 (amino acids 1673-1768) and pool 6 (amino acids 1577-1672) – were constructed using inverse PCR mutagenesis (Jain and Varadarajan, 2014) with KaPa HiFi HotStart ReadyMix. 30 base mutagenic primers were designed for each codon, with hand mixed NNN (pools 5 and 6) and NNK (pool 4) (N = ACTG, K = GT) bases at the 5′ end of the sense oligonucleotide. Ligations were pooled together, phosphorylated and self-ligated as previously described (Jain and Varadarajan, 2014). Cells were transformed into MAX Efficiency DH5α competent cells (Thermo Fisher), and 10 μl was plated on an agar plate with ampicillin (0.1 mg/mL) while the remaining 990 μl were added to 250 ml LB media with ampicillin (0.1 mg/mL). Plates were counted after 24 hr incubation and libraries with at least 10,000 colonies (approximately five-fold library coverage) were purified using the Qiagen Maxiprep kit. Pool 4 had approximately 86,000 colonies, pool 5 had 85,000 colonies, and pool 6 had 104,200 colonies. PCR (7-10 cycles) was used to isolate the mutagenized BRCA1 fragment and add a 16-base degenerate barcode to variants (primers NheI-Fwd-Long and BRCA1-AscI-Barcode-XhoI-Gib-Rev for pool 4, BRCA1-NheI-Fwd-Long and BRCA1-SbfI-Barcode-Rev for pools 5 and 6). The BRCA1 fragment was ligated back into pcDNA5-FRT/TO-HA-BRCA1 using Gibson assembly (Smith et al., 2009) and transformed into MAX Efficiency DH5α competent cells. Pool 4 had 46,300 independent plasmid variants, pool 5 had 10,400 plasmids, and pool 6 had 11,900 plasmids. Barcodes were amplified from gDNA over 18  reactions, each containing 250 ng of gDNA. PCR reactions were done using Kapa2G Robust  Polymerase for 22-23 cycles. For pool 4, the primers Pool4-Nest1-Fwd and FRT-LacZ-Rev were  used and for pools 5 and 6 the primers CTerm56-Nest1-Fwd and FRT-LacZ-Rev were used.  The resultant ~750 bp fragment was purified with 0.6X AmpurePB beads and eluted in 40 μl 10  mM Tris (pH 8). 125 ng of PCR product was re-amplified with primers containing sample  indexes and Illumina cluster-generating sequences using Kapa2G Robust Polymerase. The  resultant 150 bp (pool 4) or 170 bp (pools 5 and 6) amplicon was purified using double Ampure  purification (0.6X and 0.8X). The samples were combined, and barcodes and sample indexes  (Seq-i70x and Seq-i50x primers) were sequenced on the HiSeq 4000 at the Nationwide  Children's Hospital Genomic Services Laboratory.
GEO Accession
GSM5682066