GSM7804863: HNP RNA CrossDisorder MPRA Rep 10; Homo sapiens; OTHER
72 hours after transfection, DNA and RNA were extracted from the cells by Quick-DNA/RNA Miniprep Plus Kit (Zymo, cat#D7003) using 600uL of shield buffer and 600uL lysis buffer per well. The synthesized 200bp library oligo library was diluted to 1:20 and amplified usingMPRA-chip-primer-F and MPRA-chip-primer-R and NEBNext 2X Q5 Hifi HS Mastermix (NEB, cat#M0453S). The resulting PCR reaction was cleaned up with Zymo DNA Clean and Concentrator kit-5 (Zymo, cat#D4033). Random barcodes were added via PCR using MPRA-bc-primer-F and MPRA-bc-primer-R using NEBNext 2X Q5 Hifi HS Mastermix. The resulting PCR product was cleaned up with Zymo DNA Clean and Concentrator kit-5. The MPRA vector, Donor_eGP2AP_RC (addgene: ID#133784), and oligo library were digested with SpeI-HF (NEB, cat#R3133S) and MluI-HF (NEB, cat#R3198S). The digested oligo library and Donor_eGP2AP_RC plasmid were ligated together using T7 DNA ligase (NEB, cat#M0318S). The ligation mix was transformed into 6 reactions of Endura Electrocompetent cells (Lucigen, cat#60242-1) via electroporation. The bacteria was grown for 1 hour at 37ºC, then plated and incubated overnight on a 10 cm LB plate with kanamycin at 37ºC. The bacteria was collected by scraping and subsequently cultured in 4L of LB with kanamycin at 30ºC overnight. The LB culture was subjected to maxiprep using ZymoPURE II Plasmid Maxiprep Kit. The minimal promoter (minP) and luciferase (Luc) sequence was digested from the pMPRAdonor2 plasmid (addgene: ID#49353) using KpnI-HF (NEB, cat#R3142S) and XbaI (NEB, cat#R0145S). The band was extracted from a 1% agarose gel using Zymo Gel DNA recovery kit (Zymo, cat#D4008). The Donor_eGP2AP_RC plasmid containing the library and barcode was digested with KpnI-HF and XbaI and cleaned up with the Zymo DNA Clean and Concentrator kit-5. The digested plasmid and the minP-Luc fragment were ligated together using T7 DNA ligase. The ligase reaction was transformed into Endura Electrocompetent cells, which were plated on a 10 cm LB plate with kanamycin and grown at 37ºC overnight. The plate was scraped and grown in 4L of LB with kanamycin at 30ºC overnight. The LB culture was subjected to maxiprep using ZymoPURE II Plasmid Maxiprep Kit. (Zymo, cat#D4203). The library and barcode region was amplified off the plasmid with BC_Map_P5_R and BC_Map_P7_F_Ind_# using NEBNext 2X Q5 Hifi HS Mastermix, which added sequencing adapters and indices for demultiplexing. This PCR product was size selected and cleaned up using SparQ beads (VWR, cat#76302-834), and sequenced for barcode mapping via 2x150bp Novaseq SP with 20% Phix and custom sequencing primers: BC_Map_R1seq, BC_Map_R2seq, and BC_Map_Index. Barcodes were assigned to each oligo sequence as previously described using the custom code: https://github.com/kiminsigne-ucla/bc_map.