GSM9230322: Donor 1_Pseudo-islets ST18_KD-rep_1; Homo sapiens; RNA-Seq
Psudo-islets were collected, dissolved in Trizol, and used in a DNA-free RNA-isolation kit (Zymo R2050) for RNA purification. Total RNA preps will subject to standard Sample Quality Control analysis by using fluorometry Qubit or Picogreen and integrity by BioAnalyzer or TapeStation.  Only those with a RIN>7.2 were used for down-stream studies. For each sample, 50 ng of RNA were used for linrary preparation using the Illumina Ribo-Zero Plus rRNA Depletion kit. The procedure followed that specified by manufacturer, which include the depletion of rRNAs, cDNA reverse transcription, strand specific labeling/amplification. Library Quality Control analysis will be performed by using Qubit and BioAnalyzer to determine the concentration and size bp. Only those with significan DNA fragments between 150-1000 bp were sequenced. Each library that passes the final QC undergoes qPCR using the KAPA library quantification kit and QuantStudio. qPCR allows highly accurate quantification of library molecules that can seed clusters and balance target sample representation during pooling. Multiplexed samples are pooled in equimolar or user-defined ratios to achieve the multiplexing level requested by the user using Illumina MiSeq platform.