GSM8057770: PDXMPRA1137Rep4; synthetic construct; OTHER
MPRA Oligo design Oligo libraries were designed by following previous work with modified protocols (1-4). MPRA oligos ordered from Agilent (230 bp) were structured as follows: 5'-Primer1-enh-Kpnl-Xbal-barcode-primer2-3' where primer1 and primer2 are universal primer sites, enh denotes the 175bp variant containing region to test for enhancer activity, Kpnl and Xbal denote recognition sequences for cut sites, and barcode denotes 10-bp tag sequence (see Sup File 6). Agilent oligos were resuspended in 100 ul nuclease free water. All 10-bp barcodes for each variant allele used in MPRA are provided in Supplemental File 7. MPRA Plasmid Cloning-Input (DNA) Library construction For plasmid cloning, oligo libraries were amplified by 20 cycles of emulsion PCR (Micellula DNA Emulsion & Purification Kit #E3600, EURx Molecular Biology Products) using Herculase II fusion DNA polymerase (#600675, Agilent), forward and reverse primers (see Sup File 3) to introduce SfiI restriction enzyme sites (GGCCNNNNNGGCC) (NEB) and homology arms to the pMPRA1 plasmid (Cat: #49349, Addgene). Purified PCR products were separated on a 2-4% agarose gel to verify the expected amplification size of 281bp. The pMPRA1 backbone vector was Sfil digested overnight and size selected on a 1% agarose gel. Vector backbone gel extraction was done with the Qiagen gel extraction kit and QIAquick PCR purification kit (28706X4 and 28104).  Gibson assembly was used to clone oligos into vector using 79 ng of inserts, 100 ng of digested vector, and 20 ul Gibson assembly 2x master mix (# E2611S, NEB). The reaction was purified using MinElute PCR purification column (Qiagen), and drop analysis was performed with Millipore filters (Type VSWP 0.25 um Millipore #VSWP02500). For the transformation step, we aimed to obtain 10x CFU bacterial cells than the distinct promoter-tag combinations (unique sequences) in the oligo library. We transformed the Gibson assembly reactions into MegaX DH10B electrocompetent bacteria (#C6400-03, Invitrogen) using GenePulser II electroporator (Bio-Rad). Plasmids were extracted with Qiagen Maxi Prep Kit. For quality control studies, an aliquot of the isolated plasmid library was digested with SfiI and run on 1 % agarose gel to confirm the presence of inserts. To generate linear enhancer-barcode backbone sequences for reporter insertion, 2 ug of plasmid was digested with KpnI/XbaI. A minimal promoter + truncated eGFP was then ligated to the linearized enhancer-barcode backbone and purified using the Qiagen MilEute PCR purification kit. The ligation was then transformed into 1 vial of MegaX electrocompetent bacteria (#C6400-03, Invitrogen) as before. Plasmids were then extracted using a Qiagen Maxiprep kit as before and the elution was verified as a single size band by gel electrophoresis. MPRA library transfection and sequencing MPRA plasmid library (10 µg) transfections were done in 10 ALL cell lines having at >95% cell viability (45 million cells x 4 replicates x 10 cell lines) using electroporation with the Neon Transfection system (Thermofisher). Next day, RNA-was harvested using the RNeasy plus mini kit (Cat: #74134, Qiagen) using 4 columns per sample. Once the RNA was isolated, we performed additional DNase digestion using RQ1 RNase-free DNase (Cat: # M6101, Promega). All tubes from the same replicates were combined and added 1 volume of 70% ethanol to the combined lysate and mixed well by pipetting. The DNase treated RNA was again purified with RNeasy mini kit and eluted in 60 ul RNase free water. Total RNA was quantified using DeNovix Ds-11 FX instrument. We yielded 16µg-112µg of total RNA from each replicate depending on the cell line used. mRNA purification was performed using Dynabeads mRNA purification kit (Cat: #61006, Invitrogen). mRNA concentration was measured using Qubit HS RNA (Cat: #Q32852, Invitrogen). We yielded from 0.75-2 ug of mRNA in average from each replicate. cDNA was synthesized using three primers (2 uM) cDNA P1, cDNA P4, and cDNA 6, with the Superscript III first-strand synthesis system (Cat: #18080051, Invitrogen) (see Sup File 6). Final multiplexing of 50ng cDNA and input plasmid DNA (4 aliquots of MPRA plasmid pool that were independently prepared for next-generation sequencing) was carried out using Q5 Hot Start 2x Master Mix (NEB #M0494S), index primers, and Multiplexing primer 1 for 15 cycles of PCR. The reactions were size selected using AMPure XP beads (Cat: #A63881, Beckman Coulter, Indianapolis, IN) and eluted in 20 ul nuclease free water. The final library concentration was measured using Qubit DNA HS. 20-40 ng of each library was sequenced on the Illumina NovaSeq (200 million x 150bp paired-end reads per sample) at the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital.