GSM9039897: TetOff, dox 2 hour timepoint, rep2; Homo sapiens; OTHER
For barcode libraries derived from RNA, Trizol was used for extraction. Genomic DNA was extracted using the Qiagen Gentra Puregene Cell DNA Extraction Kit. For polysome libraries, cells were treated with 100 μg/mL cycloheximide and lysed. The lysate was run over a 15 45% (w/v) sucrose gradient to separate polysomes. RNA was extracted from each fraction. For RNA libraries and RNA extracted from polysome fractions, cDNA was generated using RevertAid Reverse Transcriptase and RT primer 5′CTCCACTTTGTACAAGAAAG. The cDNA was purified and concentrated with the Zymo DNA Clean & Concentrator Kit and all was used as template in PCRI. For DNA libraries, all genomic DNA was used in PCRI. For both assays, three PCR cycles were performed with ExTaq polymerase (primers 5′GTCAACCACCGCGGTCTC and 5′ACGACGCTCTTCCGATCTNNNNNNNNNNcactttgtacaagaaagttgaacg). Reactions were then purified with SPRIselect beads (Beckman Coulter) using a 0.7x one sided size selection protocol and used in PCRII (primers 5′CGTGTGCTCTTCCGATCTcgaccactaccagcagaaca and 5′ACACTCTTTCCCTACACGACGCTCTTCCGATCT), which was then purified with SPRIselect beads using a 0.6x to 1.2x two sided size selection protocol. A portion of this reaction was used for a final (PCRIII) reaction to add Illumina-compatible sequences (index primers from the NEBNext Multiplex Oligos for Illumina Kit). The final libraries were purified and concentrated with SPRIselect beads using a 0.5x to 1x two sided size selection protocol, quantified and submitted for Illumina sequencing.