I want to extract differential expressed genes using LIMMA from RNA seq data for three cancer types viz breast, lung and prostate. These data should have tumor and normal samples. I have read some papers which have used data from TCGA. BUt now TCGA has linked to Genomics Data Commons and all data are not open access. All BAM files are under controlled access. Also, LIMMA requires raw read counts for analysis. I an new to RNA seq data and analysis. Can anyone help, where should I get these data and what format should it be, as I have read that TPM, FPKM normalized values cannot be input to LIMMA.

Thanks in advance.

Dear Richa,

from your description, one nice package is the TCGAbiolinks R package:

https://bioconductor.org/packages/release/bioc/html/TCGAbiolinks.html

You could simply find the specific TCGA cancer projects you wish to analyze, and probably download the HTSeq raw counts-with this format, you could use any R package for downstream analysis, such as edgeR, limma + voom, etc.

For more details and the relative code pipelines, you could check their comprehensive vignette and examples.

Best,

Efstathios