title: GSM2262570: plasmid library, PCR rep2; Homo sapiens; OTHER
library_construction_protocol: To normalize SuRE expression data, we first characterized the barcode frequencies in the plasmid library. More specifically, we digested  1 µg library with I-SceI (#R0694S; NEB) in 25 µl to linearize the plasmids, then performed 2 replicate PCRs each on 2µl I-SceI digested material, using the same protocol as for the cDNA but for 8 cycles. Because of the high complexity of the library (~270 million) the aim was not to get a quantitative readout for each barcode, but rather to identify potentially over-represented barcodes and/or regions of the genome and normalize for that. The PCR material was e-gel size-selected and subjected to single-read 50 bp high throughput sequencing on an Illumina HiSeq2500 or HiSeq2000. In total we obtained ~40 million reads per PCR replicate. From these reads barcode counts were determined using cutadapt version 1.2.1 (http://journal.embnet.org/index.php/embnetjournal/article/view/200) to remove the adapter (GCTAGCTAACTATAACGGTCCTAAGGTAGCGAA) from the sequence.
attributes.tag: GEO Accession
attributes.value: GSM2262570