title: GSM9308337: K-562_pDEST-QUASARR-luc-gfp_pMYC_CNPL-TRE202-MRE_eaBC_RNA_Index1_Rep1; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted from cells using TRIzol Reagent (15596026; Invitrogen) following the manufacturer's instructions. Plasmid DNA was extracted from cells as previously described (PMID: 9591124). QUASARR-seq requires the generation of a pre-transfected DNA eaBC-TRE-paBC index library for barcode-element index generation, as well as a minimum of four libraries per replicate: two RNA libraries targeting the eaBC and paBC transcripts, respectively, and two DNA libraries targeting the eaBC and paBC inputs, respectively. To construct an eaBC-TRE-paBC index, pre-transfected eaBC-TRE-paBC libraries were generated prior to electroporation. Total RNA was extracted from cells using TRIzol Reagent following the manufacturer's instructions. To remove contaminating plasmid DNA, RNA was treated with DNase I (AM2222; Invitrogen) and subsequently purified using RNeasy Mini Kit (74104; QIAGEN). Reverse transcription was performed with the DNase-treated RNA (for eaBC and paBC quantification libraries) as the template using SuperScript III Reverse Transcriptase (18080093; Invitrogen). Plasmid DNA was extracted from cells as previously described (PMID: 9591124). A first primer extension was performed with the pre-transfected (for eaBC-TRE-paBC index library) and extracted (for eaBC and paBC quantification libraries) DNA as the templates. Reactions were treated with Exonuclease I (M0293; New England Biolabs) to remove unused primer and purified using DNA Clean & Concentrator-5 (D4013; Zymo Research Corp.). A second primer extension was performed with the products of the reverse transcription (RNA libraries) and the first primer extension (DNA libraries) as the templates, respectively. Reactions were again treated with Exonuclease I and purified using DNA Clean & Concentrator-25 (D4033; Zymo Research) and DNA Clean & Concentrator-5 for the RNA and DNA libraires, respectively. Finally, low-cycle PCR was performed to add sequencing adapters, followed by acquisition of 2 × 150 bp reads on an Illumina NovaSeq X Plus.