Plasmid library construction

All pooled plasmid libraries were constructed by Gibson-style assembly using HiFi DNA assembly master mix (NEB E2621L) and transformed using either ElectroMAX DH10B (ThermoFisher #18290015) or NEB 10-beta Competent Escherichia coli (NEB #C3019H) according to manufacturer protocol. All column purifications were performed using Zymo DNA Clean and Concentrator-5 (Zymo #D4013). Dilutions of reach transformation were plated to estimate library size and ensure sufficiency library diversity. Plasmid libraries were harvested from liquid culture using the Monarch Plasmid DNA Miniprep Kit (NEB #T1010). Miniprep kit reagents were scaled to accommodate one spin column for every 5 ml of culture.

To generate estradiol-inducible barcode-expressed plasmid libraries (fig. S1), pNTI726 and pNTI728 were first digested with BamHI. Five barcodes were generated by annealing primer RM396 with one of five barcode primers, RM391, RM392, RM393, RM394, or RM399, and extending the annealed duplex using Q5 polymerase. Four of these barcodes were inserted by Gibson assembly into BamHI-linearized pNTI726 and the last barcode DNA fragment inserted into BamHI-linearized pNTI728. Gibson reactions were purified and concentrated, transformed into ElectroMAX DH10B cells, and inoculated directly into 200 ml of lysogeny broth (LB)–carbenicillin media. Pooled libraries were harvested when batch liquid culture reached an optical density (OD) of 4.

To generate gRNA plasmid libraries, 100 pg of CRISPRi gRNA oligonucleotide library was amplified by PCR using Q5 polymerase using primers NM636 and NM637 (16). pNTI742 was digested with AvrII and the amplified guide fragments were introduced into the digested vector by Gibson assembly. The assembly product was purified and transformed into ElectroMAX DH10B cells by electroporation. Electroporated cells were inoculated directly into 500 ml of LB-carbenicillin media and harvested at an OD of 2. Serial dilutions of the initial transformation were plated to ensure sufficient library diversity (>50× coverage of 60,000 guides). Plasmid library was purified using NEB miniprep kit and pooled library was analyzed by Sanger sequencing.

Barcodes were then added to the gRNA plasmid library, targeting an average of four barcodes per gRNA (240,000 barcodes in total). First, barcode DNA fragments were generated by annealing oligos RM504 and RM505 and extending the duplex using Q5 polymerase. The following thermocycler conditions were used to avoid amplification of any specific barcode sequence. Initial denaturation at 98°C for 45 s, six cycles of annealing and extension at 68°C for 15 s and 72°C for 5 s, respectively, and finally a 12°C hold. The barcode fragments were digested with BamHI and MfeI to eliminate barcode fragments that contain these restriction sites and then purified using a DNA Clean and Concentrator-5. The library of gRNA-expressing plasmids was then digested with AflII and the barcode DNA fragments were introduced by Gibson assembly. The assembly reaction was purified using a DNA Clean and Concentrator-5 then transformed into NEB 10-beta Competent E. coli (High Efficiency) according to manufacturer protocol. Transformation dilutions were plated to estimate total number of transformants, and in parallel inoculated at several dilutions directly into LB-carbenicillin media. Liquid culture corresponding to roughly 240,000 transformants was harvested at an OD of 2 and plasmid library was extracted using NEB miniprep kit. Reagents were scaled to accommodate one spin column for every 5 ml of culture. This barcoded gRNA library was paired-end sequenced (see Barcode to gRNA assignment section) to assign barcodes with gRNAs.

P(HIS4)-yECitrine was amplified from pNTI741 using primers RM501 and RM502, P(PGK1)-yECitrine was amplified from pNTI725 using primers RM501 and RM503, and two versions of estradiol-inducible P(Z)-mCherry with specific nucleotide identifiers were amplified with either RM522 and RM524 or RM523 and RM524. The barcoded gRNA plasmid library was digested with BamHI and the four PCR products were introduced into the digested library by Gibson assembly to create the four CiBER-seq libraries. These libraries were then transformed into ElectroMAX DH10B cells by electroporation. Electroporated cells were inoculated directly into 500 ml of LB-carbenicillin media and harvested at an OD of 2. Serial dilutions of the initial transformation were plated to ensure sufficient library diversity (>50× coverage of 60,000 guides). Plasmid library was purified using NEB miniprep kit and pooled library was analyzed by Sanger sequencing.

CiBER-seq plasmid libraries for studying P(MET6), P(CWP1), P(PHO5), and P(HIS4) regulation (Fig. 1) were constructed in a similar fashion, but with key differences. pNTI743 containing divergent terminating sequences was used as the parent vector into which the gRNA library was inserted at the AvrII restriction site. Dual barcodes were next inserted into this gRNA plasmid library by digesting the plasmid library with AscI, annealing and extending RM720 and RM721, and Gibson assembling. Finally, the divergent promoters containing P(PGK1)-citrine normalizer and P(query)-mCherry expression cassette were inserted in between the two barcodes by: (i) digesting pNTI757 through pNTI760 with BciVI and gel extracting divergent promoter template; (ii) digesting dual barcoded gRNA plasmid library with AscI which cuts in between the two barcodes; and (iii) Gibson assembling each divergent promoter into the barcoded gRNA plasmid library and separately transforming and preparing these plasmid libraries as outlined above.