title: GSM6047560: Txnrd1 KO GFP.3; Mus musculus; RNA-Seq
library_construction_protocol: Pancreatic islets were isolated from experimental mice (age 10-20 weeks) and cultured overnight at 37 degrees C, 5% CO2, in CMRL supplemented with 10% heat-inactivated FBS and containing 5.5 mM glucose. The next day, islets were dispersed into single cells using trypsin. Populations of live GFP+ (beta cells) and Tom+ (non-beta cells) were collected by fluorescence-activated cell sorting (FACS). DAPI was used to exclude dead cells. Total RNA was isolated using an RNeasy kit (Qiagen). Genomic DNA was removed using a TURBO DNA-free Kit (ThermoFisher Scientific). RNA was quantifed by fluorimetric methods (Qubit, Invitrogen) and quality assessed by Fragment Analysis (Agilent).  cDNA was synthesized using 5-10ng of input RNA with with libraries prepared following manufacturer's recommendations in the SMARTseq Ultra Low Input workflow (Takara Bio Inc.).  Libraries were completed, indexed and amplified with the Nextera DNA Library kit (Illumina).  Final libraries were quantified by qPCR (Kapa Library Quantification Kit, Kapa Biosystems) and fragment size observed with high sensitivity DNA fragment analysis (Agilent).  Paired end sequencing was completed on the NovaSeq 6000 at 2x100bp reads, targeting 50 million reads per sample.