title: GSM7328834: EoE/AD MPRA Jurkat 5; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted by the RNeasy Midi Kit following the manufacturer's instruction. Extracted RNA was subjected to DNase treatment. eGFP mRNA enrichment and elution were performed using biotinylated probes as in Lu et al (PMID: 33712590), and the mRNA was reverse transcribed to cDNA using SuperScript™ IV First-Strand Synthesis System with gene specific primer MPRA_v3_Amp2Sc_R, following the manufacturer's instruction. We assembled the MPRA libraries as in Lu et al  (PMID: 33712590), with minor modifications. A total of two versions of the library were assembled. The first version was built following the protocol in Lu et al (PMID: 33712590) and used in TE-7 related experiments. For the second version, an additional step was added after the assembled backbone library was transformed into Escherichia coli (E. coli) through electroporation. Electroporated E. coli were immediately split into eight 1 mL aliquots of SOC medium and recovered for 1 hour at 37°C. For each of the aliquots, serial dilutions of recovered SOC medium were plated to estimate the number of colony forming units. Based on the estimation, volumes enough for 50x, 100x and 200x of the allele number in the oligo pool were independently expanded in 100 mL of LB Broth supplemented with 100 μg/mL of carbenicillin at 37 °C for 12–16 h. Plasmids were then extracted using the QIAGEN Plasmid Maxi Kit. The oligo and barcode regions were amplified from three backbone libraries to build sequencing libraries as described in Lu et al  (PMID: 33712590). Samples were purified, molar pooled, and sequenced using 2 × 150 bp on Illumina NovaSeq 6000. After oligo and barcode association analysis,  the backbone library with ~200x coverage was selected for building the transfection library.  This second version was used in HaCaT and Jurkat related experiments.