GSM7505984: Co-recruit library screen, repression screen, bound R1; Homo sapiens; OTHER
Genomic DNA was extracted with the QIAgen Blood Maxi Kit following the manufacturer?s instructions with up to 1 × 108 cells per column. Domain sequences were amplified by PCR with primers containing Illumina adapters as extensions. 27-55x 100uL PCRs were set up on ice, with the genomic DNA available for each experiment dictating the number of reactions. 10 ug of genomic DNA, 0.5 uL of each 100M primer, and 50uL of NEBnext Ultra 2x Master Mix (NEB) was used in each reaction. Samples were thermocycled at 98C for 3m; 33 cycles of 98C for 10s, 63C for 1m, 72C for 30s; and 72C for 2m. PCR reactions were pooled and run on a 1% TAE gel, the 600bp band was excised, and DNA was purified using QIAquick gel extraction kit (Qiagen) and eluted into nonstick tubes (Ambion). Samples were sequenced on an Illumina HiSeq (2×150bp, Admera Health). Library members were chosen from the Nuclear Pfam library described in21. A total of 20 negative control domains, 10 randomers and 10 tiles of the DMD protein; 30 activator domains; and 50 repressor domains were chosen for cloning. Of the 50 repressors, 6 were discovered to have errors introduced during design, and were discarded from downstream analysis. All domains, including those eventually discarded, are listed in Supplementary Table 1. After library assembly, DNAChisel77 was used to optimize coding sequences by removing duplicates, 7xC homopolymers, BsmBI and BbsI restriction sites, and rare codons. Codon usage was matched to human codon prevalence and GC content was restricted to be between 20% and 75% in any 50-nucleotide window and between 25% and 65% globally. Cloning for the combinatorial screen proceeded in two stages; in the first, domain-linker-domain concatenations were assembled, and in the second, concatenations were placed into a lentiviral backbone. Domains in the N-terminal and C-terminal position were synthesized as 2 separate oligonucleotide pools (Twist Biosciences). Each pool was PCR amplified in a clean PCR hood to avoid DNA contamination. Each pool was split into 6x 50 uL reactions that were PCR amplified for 21 cycles with 5 ng template, 1 uL of each 10mM primer, 1uL of Herculase II polymerase (Agilent), 1 uL of DMSO, 1 uL of 10mM dNTPs, and 10 uL of 5x Herculase buffer (Agilent). Reaction mixes were thermocycled at 98C for 3m; then 21x cycles of 98C for 20s, 61C for 20s, and 72C for 30s; and, finally, 72C for 3m. Reaction products were pooled and gel extracted by loading a 1% TAE gel, excising the 300 bp band, and purifying using a Zymo Research gel extraction kit. Each of the 2 extraction products (N-terminal and C-terminal) were then amplified for 23x cycles each using the same protocol in order to generate sufficient DNA for downstream reactions. To generate concatenations, 5 uL of 15 ng/uL product from each product, N-terminal and C-terminal, were mixed with 4 uL T4 buffer (NEB B0202S), 4 uL BbsI-HF (NEB R3539L, 1 uL T4 ligase (NEB M0202M), and 1uL of an XTEN linker-encoding DNA amplicon. This linker was encoded with variable nucleotides in codon wobble positions to permit variable DNA sequence in between domains and prevent lentiviral recombination without varying the amino acid sequence of the linker. To complete the GoldenGate reaction, the mix was thermocycled for 65x cycles at 37C for 5m, then 16C for 5m, before a series of incubations at 37C for 1h, 65C for 20m, and 16C for 1h. The reaction product was run on a 1% TAE gel and the 600bp band of domain pair concatenations was extracted with a Zymo gel extraction kit. The concatenation product was then PCR amplified as before, using the outer primers, for 15x cycles. Amplification products were then split into 12x GoldenGate reactions, each consisting of 75ng (1.33 uL) Bsmbi-v2 digested pJT039 lentiviral backbone, 10 ng (0.64 uL) concatenation amplicon, 2 uL T4 buffer, 1 uL BsmbI-v2 Golden Gate Assembly Kit (NEB E1602L), and 15.03 uL nuclease free H2O. Reactions were thermocycled with 65x cycles at 42C for 5m and 16C for 5m, before a final incubation at 42C for 5m and 70C for 20m. The reactions were then pooled and purified with a QIAgen MinElute column, eluting in 6 uL ddH2O. 2 uL of eluent was then electroporated into each of 2 tubes of 50 uL Endura electrocompetent cells (Lucigen, Cat#60242-2) as per manufacturer?s instructions. Cells were then plated onto 4x large 10? x 10? LB plates with carbenicillin. After overnight growth colonies were scraped into a collection bottle and plasmid pools were extracted using a plasmid maxiprep kit (Qiagen #12662). 2 smaller plates of LB with carbenicillin were also prepared with 1:20 diluted cells to count colonies and confirm transformation efficiency. Approximately 50 colonies were Sanger sequenced (Quintara) to estimate cloning efficiencies and the proportion of empty backbone plasmids in the pool.