GSM2102061: KLF5+/+; Vil-CreERT2 Sample 2; Mus musculus; RNA-Seq
Jejunal tissue was extracted and flushed of excrement then cut into pieces.  Tissue was then subjected to a 30 minute incubation in 2mM EDTA in PBS at 4C.  Tissue was then transferred to 5mL shaking buffer (43.3mM sucrose, 54.9mM sorbitol in PBS).  Tissue was shaken for 1 - 2 minutes until crypts were visible under the microscope.  After crypts were isolated, the buffer with chelated tissue was poured through a 70uM filter to remove villi.  RNA was then prepped using the Qiagen RNeasy kit. A double-stranded DNA library was created using 250ng of total RNA, quantitated by picogreen, preparing the fragments for hybridization onto a flowcell. cDNA was created using the fragmented 3’ poly (A) selected portion of total RNA and random primers. Libraries were created from the cDNA first blunt ending the fragments, attaching an adenosine to the 3’ end, and ligating unique adapters to the ends. The ligated products were then amplified using 15 cycles of PCR. The resulting libraries were quantitated using NanoDrop and fragment size assessed with the Agilent 2100 Bioanalyzer. A qPCR assay was performed determine the concentration of adapter ligated fragments using the Applied Biosystems ViiA 7 instrument and a KAPA Library Quant Kit.
GEO
GSM2102061