title: GSM6745677: 455 MPRA experiment on the mouse liver (3prime library, enhancer-barcode assignments); synthetic construct; OTHER
library_construction_protocol: Enhancer library cloning: The pSA293-CHEQseq plasmid (Addgene 174669), containing a SCP1 promoter, a chimeric intron and the Venus cDNA, was used as a reporter plasmid for MPRA. The 455 library was cloned in a newly generated pSA293-CHEQseq-3'BC-2 containing an 18 bp BC optimized for ONT sequencing with the following pattern: NNNYRNNNYRNNNYRNNN. The oligonucleotide libraries were resuspended according to the manufacturer's recommendation and amplified via PCR with the primers 'CHEQ_liver_For' and 'CHEQ_liver_Rev'. To clone the amplified enhancer library upstream of the SCP1 promoter, the vectors were linearized via inverse PCR with primers 'CHEQ_lin_For' and 'CHEQ_lin_Rev'. Amplified libraries and the corresponding linearized vector were combined in an NEBuilder reaction with a vector to insert ratio of 1:5. The NEBuilder reactions were dialyzed against water in a 6 cm Petri dish with a membrane filter MF-Millipore 0.05 µm (Merck, Kenilworth, NJ) for 1 hr. Reactions were recovered from the membrane, and 2.5 µL of the reaction was transformed into 25 µL of Lucigen Endura ElectroCompetent Cells (Biosearch Technologies, Hoddesdon, UK). Before culture for maxiprep, 1:100,000 of the transformed bacteria was plated on an LB-agar dish with carbenicillin to estimate the complexity of the cloned library. A volume of bacteria corresponding to a complexity of 500 BCs per enhancer was put in culture for maxiprep. Maxiprep was performed with the Nucleobond Xtra endotoxin-free maxiprep kit (Macherey-Nagel). Bulk MPRA in vitro: The MPRA libraries were transfected in HepG2 cells by use of the Lipofectamine 3000 reagent (Thermo Fisher Scientific). Briefly, 4 million HepG2 cells were seeded in a 10 cm cell culture dish. The next day, when cells reach 70-90% confluency, a tube A with 500 µL opti-MEM (Thermo Fisher Scientific) and 25 µL Lipofectamine 3000 reagent and a tube B with 500 µL opti-MEM and 15 µL of the liver enhancer library were prepared and incubated for 5 min at RT. Tube B was mixed carefully with tube A and incubated for 15 min at RT. The medium of the HepG2 cells was also changed to opti-MEM medium and finally the mixture was added dropwise to the cells. 48 hours post-transfection, cells were detached from the plate using trypsin (Thermo Fisher Scientific). One-fifth of the cells was used for plasmid DNA extraction (Qiagen). The remaining cells were used for RNA extraction using the innuPREP RNA Mini Kit 2.0 (Analytik Jena), followed by mRNA isolation using the Dynabeads mRNA purification kit (Ambion) and cDNA synthesis using the GoScript RT Kit and oligo dT primer (Promega). To amplify the random 5' and 3' BC from the plasmid DNA or cDNA sample, a PCR was performed for 16 cycles with 'CHEQseq_barcode_5'_For', 'CHEQseq_barcode_5'_Rev' and 'CHEQseq_barcode_3'_For', 'CHEQseq_barcode_3'_Rev', respectively. To add Illumina sequencing adaptors, all samples were finally amplified by PCR for 6 cycles with the primers 'i5_Indexing_For' and 'i7_Indexing_Rev. Bulk MPRA in vivo: For intrahepatic delivery of the liver MPRA libraries, 8- to 10-week-old mice were secured and hydrodynamically injected with 20 µg of the libraries via the lateral tail vein. All libraries were diluted in sterile filtered 0.9% NaCl, and the total volume was adjusted to 10% (in mL) of the total body weight (in grams). 24 hours post-injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50 mg/kg) and whole livers were isolated. Liver tissues were homogenized by using M tubes (Miltenyi Biotec) and the GentleMACS Dissociator (Miltenyi Biotec). RNA and plasmid DNA extraction, mRNA purification and cDNA prep, and barcode amplification, were done as described for MPRA in vitro. FACS MPRA in vivo: For intrahepatic delivery of the liver MPRA libraries, 8- to 10-week-old mice were secured and hydrodynamically injected with 20 µg of the libraries via the lateral tail vein. All libraries were diluted in sterile filtered 0.9% NaCl, and the total volume was adjusted to 10% (in mL) of the total body weight (in grams). 48 hours post-injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50 mg/kg). Livers were perfused for 5 minutes with 40 mL of perfusion medium SC-1 (8 g/L Nacl, 400 mg/L KCl, 75.5 mg/L NaH2PO4, 120.5 mg/L Na2HPO4, 2,38 g/L HEPES, 350 mg/L NaHCO3, 190 mg/L EGTA, 900 mg/L D-(+)-Glucose, 1.2 mL Phenol Red solution) to remove the blood, followed by perfusion with 40 mL of SC-2 medium (8 g/L Nacl, 400 mg/L KCl, 75.5 mg/L NaH2PO4, 120.5 mg/L Na2HPO4, 2,38 g/L HEPES, 350 mg/L NaHCO3, 560 mg/L CaCl2.2H2O, 1.2 mL Phenol Red solution) containing 10 mg of collagenase P (Merck) for 5 minutes. Each lobe was dissected off and minced into small pieces in a beaker containing 39 mL SC-2 supplemented with 1 mL DNase I (Sigma-Aldrich) and 20 mg collagenase P, followed by rotating incubation for 15 min at 37°C. Hepatocytes were centrifuged for 2 min at 50 g, washed with PBS, centrifuged again for 2 min at 50 g, resuspended in 3 ml Hoechst buffer (DMEM + 10% FBS + 10 mM HEPES) and filtered through a 70 µm strainer. The protocol for hepatocyte staining was adapted from Ben-Moshe S. et al. After counting the cells on a LUNA cell counter, the concentration was adjusted to 5 million cells in 1 mL of Hoechst buffer. To determine the ploidy of hepatocytes, DNA was stained with Hoechst (Thermo Fisher Scientific) (15 µg/mL). Reserpine (5 µM) was also supplemented to prevent Hoechst expulsion from the cells. Cells were incubated for 30 min at 37°C. Hepatocytes were centrifuged for 5 min at 1,000 rpm at 4°C and the supernatant was discarded. Cells were resuspended in cold PBS in a concentration of 1 million cells in 100 µL. After spinning down (1,000 rpm for 5 min at 4°C), cells were resuspended in FACS buffer (2 mM EDTA, pH 8, and 0.5% BSA in 1x PBS) at a concentration of 1 million cells in 100 µL. Cells were stained with the following antibodies (BioLegend) at a dilution of 1:300: PE anti-mouse/human CD324 E-cadherin (catalogue no. 147304) and APC anti-mouse CD73 (catalogue no. 127210). FcX blocking solution (BioLegend catalogue no. 101319) was added at a dilution of 1:50. Cells were sorted by FACS-Aria-Fusion (BD Biosciences) using a 100 µm nozzle. FSC-A and SSC-A were used for hepatocytes size selection. Cells containing the library were selected based on GFP. Tetraploid hepatocytes were selected based on Hoechst stain. CD73 and Ecad were used to select hepatocytes bins along the porto-central axis, obtaining 100,000-200,000 cells per bin. RNA extraction, mRNA purification and cDNA prep were done as described for MPRA in vitro. To amplify the enhancer barcode on the cDNA small modifications were made. To amplify the random 3' BC, a PCR with 24 cycles was performed. To add Illumina sequencing adaptors, a PCR with 10 cycles was done. For the liver libraries cloned in the pSA293-CHEQseq-3'BC plasmids, we performed Nanopore sequencing as follows. A total of 1.5 µg of the library was linearized by digestion with NcoI according to the manufacturer's protocol. We next processed 200 ng of the cleaned up and linearized plasmid with an Oxford Nanopore Technologies Q20+ ligation sequencing kit early-access SQK-LSK112 following the manufacturer's protocol (Genomic DNA by Ligation; revision GDE_9141_v112_revC_01Dec2021). A total amount of 10 fmol of the prepared library was loaded onto a MinION R10.4 flow cell and sequenced for at least 72 hr (MinKNOW ≥ v21.11.09). All cDNA/Plasmid libraries were sequenced on a NextSeq2000 instrument (Illumina) with the following sequencing parameters: 51 bp read 1 – 8 bp index 1 – 8 bp index 2 – 51 bp read 2.