GSM5765510: ctrl crypt rep3; Mus musculus; RNA-Seq
Intestinal fragments were incubated 30 min at 4ºC in 10mM EDTA after which they were transferred to BSA 0,1% in PBS and vortexed between 30 s and 1 min. The supernatants containing the epithelial cells were filtered with a 70mm cell strainer. At this step, crypts went through the cell strainer and villi were retained on it. Crypt and villi fractions were then centrifuged separately and the pellets were frozen in liquid nitrogen until processed. Total RNA library preparation and sequencing were performed by Novogene Co., Ltd, as a lncRNA sequencing service, including lncRNA directional library preparation with rRNA depletion (Ribo-Zero Magnetic Kit), quantitation, pooling and PE 150 sequencing (30G raw data) on Illumina HiSeq 2500 platform. Filtering and trimming of the RNA seq data left around 230-300 million reads pairs /sample for mapping to the mouse reference genome, with a Pearson correlation coefficient (R2) between biological replicates samples around 0.9, ensuring a reliable expression.