RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

For the cell lines, RNA was extracted from the cells using the RNEasy® kit (Qiagen), generating high‐quality total RNA (i.e., RIN > 8) that was used as input material for library construction with Illumina TruSeq Stranded mRNA reagents. The samples were sequenced on the Illumina HiSeq2500 platform to a depth of ~20 million reads. Raw sequences were mapped to the human reference genome GrCh38 and further quantified using the Kallisto software (Bray et al, 2016). TPM values for genes were generated by summing up TPM values for the corresponding transcripts generated by Kallisto. All cell line data are available at http://www.ncbi.nlm.nih.gov/bioproject/PRJNA183192.

Procedures for extraction of RNA from tissues, library preparation, and sequencing have been described elsewhere (Uhlén et al, 2015). Briefly, reads were mapped to the human reference genome assembly GRCh38 and quantified using Kallisto version 0.42.4. Normalized expression levels (TPM values) on gene level were obtained by summing the estimated values from the constituent transcripts of each gene, respectively. All tissue data are available at http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1733/.

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Manufacturer protocol

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