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Pipeline publication

[…] itrogen, 15140122), 0.5 μg/ml hydrocortisone (Sigma, H-0888), 100 ng/ml cholera toxin (Sigma, C-8052), 10 μg/ml insulin (Sigma, I-1882), and 20 ng/ml recombinant human epidermal growth factor (Peprotech, 100–15) as previously described []. MCF-7 cells were obtained from ATCC and were cultured in DMEM supplemented with 10 % fetal bovine serum and penicillin/streptomycin., The RNA-seq libraries were generated with TruSeq Stranded Total RNA with Ribo-Zero Gold Kit and the samples were sequenced as 100-bp single-end reads using a Hi-Seq 2000 instrument. For the RNA-Seq analysis, the adapter sequences were first removed from the RNA-seq reads. Ribosomal RNA reads, if any, were filtered out using Bowtie []. After quality filtering and adapter removal steps, the reads were aligned to a transcriptome and quantified using RSEM v.1.2.7 []. The annotation file was downloaded from University of California, Santa Cruz (UCSC) genome browser, human hg19 assembly. To quantify gene expression, gene counts and transcripts per million (TPM) were calculated by using the RSEM tool. Differential gene expression was calculated using the Deseq2 version 1.4.5 package in R 3.1.0 using the mean value of gene-wise dispersion estimates []. To find significant differentially expressed genes, we used 0.01 for adjusted p value and >1 log2 fold change. Gene ontology analysis was performed with the FuncAssociate software []. The RNA-seq plots were confirmed using the ngs.plot software []., Hi-C was performed as previously described with minor modifications []. The modified part of the protocol was in the biotin incorporation step, where the mixture was incubated at 37 °C for 40 minutes with continuous shaking and tapping of the tube every 10 minutes. The MCF-10A and MCF-7 Hi-C samples displayed a range of 40–85 % biotin incorporation efficiency. At the end of Hi-C sample preparation, the libraries were sequenced using PE100 reads with a Hi-Seq 2000 instrument., Figure S1 in Additional file summarizes the mapping results and different classes of reads and interactions observed []. The data were binned at 6.5-Mb, 1-Mb, 250-kb, 100-kb, and 40-kb non-overla […]

Pipeline specifications

Software tools Bowtie, RSEM, DESeq2, FuncAssociate, ngs.plot
Diseases Breast Neoplasms, Breast Diseases, Neoplasms