Illumina HiSeq 4000 paired end sequencing; RNAseq analysis of Chrnb4-cre driven Dicer conditional knockout (Dicer CKO) whole retinas
RNAseq analysis of Chrnb4-cre driven Dicer conditional knockout (Dicer CKO) whole retinas
The neural retinas (NR) of enucleated eyes were dissected in 1X PBS (with no RPE). The NR were then placed in 1.5ml eppendorf tubes in 1X PBS and were span down. The supernatant (PBS) was discarded. RNA was either extracted straight away or the NR were frozen down in dry ice. Once frozen, they were stored at -80ºC for long term storage.Total RNA extraction from NR was carried out using the miRNeasy Micro Kit from Qiagen (217084), following manufacturers instructions. An optional DNase step (Qiagen 79254) was also included in the protocol, following manufacturers instructions, in order to remove genomic DNA. RNA was quantified using an Agilent TapeStation (TapeStation Analysis Software A.02.01) for RNA sequencing. Extracted RNA was stored at -80ºC before sequencing. RNA was extracted using the miRNeasy Micro Kit (Cat No./ID: 217084) following manufacturer's instructions and including the optional DNase step. PolyA- Illumina's TruSeq stranded kit (RS-122-2103) was used for library prep.