Thus, at face value, the non-small-cell lung carcinoma layered effect of dose-responsive compensation and feed-forward dosage compensation may explain all of the final increase in S2 cell X chromosome expression (1.50-fold��1.35-fold=2.03-fold). While most work on dosage compensation focuses on the X chromosome ,, other organisms also show dosage compensation on autosomes . For example, mammalian trisomies show only about a 1.3-fold increase in gene expression as a result of a 1.5-fold change in gene dose ,. Compensation is likely to be a universal property of biological systems that enables cells to avoid deleterious effects of genetic load and other perturbations. Materials and Methods Cell Strains and Media Drosophila S2 cells  (a.k.a.
SL2) were obtained from Drosophila RNAi Screening Center (DRSC, Harvard Medical School, Boston, MA) and were grown at 25��C in Schneider’s Drosophila Medium (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine serum (SAFC Biosciences, Lenexa, KS) and Penicillin-Streptomycin (Invitrogen, Carlsbad, CA). These cells were used for all experiments, except CGH, where S2-DRSC cells were obtained from the Drosophila Genomics Resource Center (#181, Bloomington, IN). Sequencing We extracted S2 cell genomic DNA using a genomic DNA kit (Qiagen, Valencia, CA). Approximately 2 ��g of purified genomic DNA was randomly fragmented to less than 1,000 bp by 30 min sonication at 4��C with cycles of 30 s pulses with 30 s intervals using the Bioruptor UCD 200 and a refrigerated circulation bath RTE-7 (Diagenode, Sparta, NJ).
Sonicated chromatin (see ChIP protocol) was purified by phenol/chloroform extraction. We extracted S2 cell total RNA with Trizol (Invitrogen, Carlsbad, CA) and isolated mRNA using Oligotex poly(A) (Qiagen, Valencia, CA). The number of cells used for each extraction was counted using a haemocytometer. The quality of mRNA was examined by RNA 6000 Nano chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA) according to the manufacture’s protocol. One hundred ng of the extracted mRNA was then fragmented in fragmentation buffer (Ambion, Austin, TX) at 70��C for exactly 5 min. The first strand cDNA was then synthesized by reverse transcriptase using the cleaved mRNA fragments as template and high concentration (3 ��g) random hexamer Primers (Invitrogen, Carlsbad, CA).
After the first strand was synthesized, second strand cDNA synthesis was performed using 50U DNA polymerase I and 2U RNaseH (Invitrogen, Carlsbad, CA) at 16��C for 2.5 h. Deep sequencing of both DNA and short cDNA fragments Dacomitinib were performed ,. Libraries were prepared according to instructions for genomic DNA sample preparation kit (Illumina, San Diego, CA). The library concentration was measured on a Nanodrop spectrophotometer (NanoDrop products, Wilmington, DE), and 4 pM of adaptor-ligated DNA was hybridized to the flow cell.