title: GSM4085061: INS1 832/13 RNA_rep3; Rattus norvegicus; OTHER
library_construction_protocol: We electroporated 10 ug of barcoded STARR-seq library into 25 million the 832/13 rat insulinoma cell line for each biological replicate (3 replicates), and harvested the cells twenty-fours later. We isolated total RNA using TRIZOL reagent (Life Technologies) following the manufacturer's protocol up to phase separation. After phase separation, we transferred the aqueous phase of the solution to a new 1.5 mL Eppendorf tube, added 1:1 volume of 100\% ethanol, and then column-purified using the Direct-zol RNA Miniprep Kit (Zymo) following the manufacturer's protocol. We further purified mRNA using Dynabeads oligo(dT) beads (Thermo Fisher). We treated 2 ug of mRNA with RNase-free DNaseI (invitrogen) to eliminate possible plasmid DNA contamination, and then reverse transcribed it into cDNA using the SuperScript III First-Strand Synthesis kit (invitrogen) with a custom primer that specifically recognizes 'STARR transcripts' (i.e. mRNA that had been transcribed from the STARR-seq plasmids). To eliminate any residual plasmid contamination in cDNA samples, we treated cDNA with DpnI (NEB), and purified the reaction using the DNA Clean and Concentrator-5 kit (Zymo). We constructed Illumina sequencing library via two serial rounds of PCR. In the first round, we used a primer set to specifically amplify STARR transcripts using cDNA as starting material. In the second round, we used a primer set to append the P5/P7 Illumina sequences using the PCR product from the first round as starting material. In both rounds, we PCR-amplified the fragments until the amplification curve reached a mid-log phase, and then purified the products for subsequent steps using the DNA Clean and Concentrator-5 kit (Zymo). To identify the barcodes corresponding to each CAGE element in the STARR-seq plasmid, 1ng of each library constructed was used in a polymerase chain reaction with primers flanking the allele and the barcode to generate fragments which were subsequently gel verified and extracted using Zymo gel extraction kit (Zymo). 25ng of the purified product was subjected to self-ligation at 16 °C overnight in a total volume of 50uls and column purified using Qiagen (Qiagen) PCR purification kit as per manufacturers recommendations. The purified fragments were subsequently treated with 10U of Plasmid-Safe ATP-Dependent DNase for 1 hour in the presence of 25mM ATP to remove unligated linear DNA fragments. 1ul of the recircularized fragments were subjected to another round of PCR resulting in a smaller fragment. Briefly an aliquot of this product was diluted 1:10 in DNAse/RNase free water and amplified in a PCR reaction, with Illumina P5 and P7 adapters, until saturation to generate libraries. The libraries were subsequently column purified, quantified and sequenced.