RNAseq of 92.1 treated with dox
Cells were plated in 6-well plates and INPP5A knockdown was induced by the addition of 100 ng/mL of doxycycline. At indicated time point, 350 L RLT buffer supplemented with 1% -Mercaptoethanol was added to each well, and then cells were scrapped on ice. Cell suspensions were directly transferred to eppendorf tubes and stored at -80C for lysis completion. Cell lysates were subsequently homogenized using QIAshredder spin columns (QIAGEN, cat. no. 79656). Total RNA was isolated using RNeasy Kit (QIAGEN, cat. no. 74104) according to the manufacturers protocol. RNA quality and concentration were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 BioAnalyzer (Agilent). 200 ng RNA (RNA Integrity Number (RIN) > 9.0) was used as input for the library preparation. RNA-seq libraries were generated using the TruSeq RNA Sample Prep Kit v2 (Illumina) according to the manufacturers recommendation. Next-generation sequencing was performed on a HiSeq2500 (Illumina).