GSM1207477: EStTA-PB-B-21, mapping restr enz 2; Mus musculus; OTHER
Total RNA was isolated from each of the clones using Qiagen RNeasy kit.  Genomic DNA was isolated using Qiagen DNeasy kit. Mapping of the barcoded PB insertion sites was done by inverse PCR coupled with high throughput sequencing. Briefly, 2 μg of gDNA was digested with 20 units of DpnII (New England Biolabs) overnight at 37°C in a volume of 100 μl. Subsequently, 600 ng of purified digested DNA was self-ligated with 40 units of high concentration T4 DNA ligase (Promega) overnight at 4°C in a volume of 400 μl. The ligation reactions were phenol/chloroform/isoamylalcohol extracted and ethanol precipitated. DNA pellets were dissolved in 30 μl of water. 5 μl of each sample was used as a template for amplification of fragments containing both the barcodes and flanking genomic DNA regions. PCR was performed in three rounds and purified products were directly used for high throughput Illumina paired-end sequencing. To measure the barcode expression levels (expression_counts), 2 μg of total RNA was reverse transcribed in a 50 μl reaction containing 50 ng of oligo(dT) primer and 1 μl of Superscript II (Invitrogen). 1 μl of cDNA was used as a template for amplification of barcode sequences . PCR was performed in two rounds and purified products were directly used for high throughput Illumina single-read sequencing. To quantify the barcode abundances for normalization (normalization_counts), 100 ng of gDNA instead of cDNA was used as a template.