GEO help: Mouse over screen elements for information.
|Public on Oct 01, 2016
|hepatocellular carcinoma cell line
|cell line: HepG2
cell type: hepatocellular carcinoma cell line
|For the lentiMPRA, 2.4 million HepG2 cells were plated on a 10 cm dish and cultured for 24 hours. The cells were infected with integrating or non-integrating lentivirus libraries along with 8 ug/ml polybrene, and incubated for 4 and 3 days, when they have an estimated 50 and 100 viral particles/cell, respectively. Three independent replicate cultures were infected.
|HepG2 cells (ATCC) were maintained in DMEM supplemented with 10% FCS, glutamine (2 mM), penicillin (100 U/ml) and streptomycin (50 μg/ml)
|The cells were washed with PBS three times, and genomic DNA and total RNA was extracted using AllPrep DNA/RNA mini kit (Qiagen). Messenger RNA (mRNA) was purified from the total RNA using Oligotex mRNA mini kit (Qiagen) and treated with Turbo DNase to remove contaminating DNA.
For each RNA replicate, 3x500ng was reverse transcribed with SuperscriptII (Invitrogen 18064-014) using a primer downstream of the barcode, which contained a sample index and a P7 Illumina adaptor sequence. The resulting cDNA was pooled and split into 24 reactions, amplified with Kapa Robust polymerase for 3 cycles using this same reverse primer paired with a forward primer complementary to the 3’ end of EGFP with a P5 adaptor sequence (BARCODE_lentiF_v4.1, Table S3). The implemented two-round PCR set-up is supposed to reduce PCR jack-potting effects and allows for incorporating unique molecular identifiers (UMIs), which could be used to correct for other PCR biases in future experiments. PCR products are then cleaned up with AMPure XP beads (Beckman Coulter) to remove the primers and concentrate the products. These products underwent a second round of amplification in 8 reactions per replicate for 15 cycles, with a reverse primer containing only P7. All reactions were pooled at this point, run on an agarose gel for size-selection, and submitted for sequencing. For the DNA replicates, 16x500ng of each replicate was amplified for 3 cycles just as the RNA. First round products were cleaned up with AMPure XP beads, and amplified for another 16 reactions, each for 20 cycles. Reactions were pooled, gel-purified, and sequenced.
|Illumina NextSeq 500
|Targeted barcode amplification from DNA
|Library strategy: lentivirus-based massively parallel reporter assay (lentiMPRA)
Libraries were sequenced on MiSeq or NextSeq 500 instruments (Illumina) and base called using the instruments' Real Time Analysis softwarre. Multiplexed paired end (PE) Illumina sequencing data was split based on sample barcodes allowing up to one substitution in the barcode sequence.
Paired-end reads shorter or equal to read length were consensus called and adapter trimmed.
For oligo validation, consensus sequences were aligned to all designed oligo sequences using BWA MEM (Li and Durbin 2009) with parameters penalizing soft-clipping of alignment ends (-L 80). For RNA/DNA counts, sequences were matched against the designed barcode sequences.
To normalize RNA and DNA for different sequencing depths in each sample, we divided reads by the sum of observed counts and reported them as counts per million. Only barcodes observed in RNA and DNA of the same sample were considered.
To determine the RNA/DNA ratios per insert, we summed up the counts of all barcodes contributing and determined the ratio of the average normalized counts.
Supplementary_files_format_and_content: All processed data files are in gzip-compressed tab-separated text file format. DNA.tsv and RNA.tsv files contain assigned RNA and DNA barcode counts. ActivityRatios.tsv is contains RNA/DNA ratios as well as all annotation values explored in the study. ColumnDescriptionActivityRatios.tsv provides a description for the short column labels used in ActivityRatios.tsv. validationExpDesignBarcodes.tsv contains the barcode counts observed in the design validation experiment. liverEnhancer_design.tsv contains all designed oligo sequences.
|Jun 30, 2016
|Last update date
|May 15, 2019
|University of Washington
|3720 15th Ave NE
|A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity