title: GSM2913924: Normal colon organoids on differentiation medium STAR+; Homo sapiens; RNA-Seq
library_construction_protocol: RNA from the FACS sorted organoids was extracted using the RNeasy RNA extraction kit (Qiagen) with DNAseI treatment. A maximum of 250ng RNA per sample was used for ribosomal RNA depletion using the Ribo-Zero Gold rRNA removal kit (Illumina) followed by an ethanol precipitation. Afterwards the RNA was fragmented for 5 minutes at 95C using 5x fragmentation buffer (200mM tris-acetate, 500mM potassium acetate, 150mM magnesium acetate, pH 8.2). Ethanol precipitation was used to precipitate the fragmented RNA followed by first strand cDNA synthesis with Superscript III (Life Technologies) and second strand cDNA synthesis. Samples were purified using QIAquick MinElute columns (Qiagen) with ERC buffer. A maximum of 2ng cDNA per sample was used for library preparation using the Kapa Hyper Prep Kit (KAPA Biosystems). In short, end repair and A-tailing of the double stranded cDNA was done by incubating the cDNA with end repair and A-tailing buffer and enzyme mix followed by incubation at 20C for 30 minutes and 65C for 30 minutes. Afterwards, unique NEXTflex DNA barcodes (Bioo Scientific) were ligated to the samples by incubation with ligation buffer, DNA ligase, and the NEXTflex DNA barcode (14nM) for 15 minutes at 20C. The barcode ligated samples were purified using a 0.8x SPRI magnetic beads cleanup (Agencourt AMPure XP reagent). The purified samples were incubated for 15 minutes at 37C with USER enzyme (New England Biolabs) to digest the 2nd cDNA strand. Libraries were amplified by adding 2x KAPA HiFi Hotstart ReadyMix and 10x Library Amplification Primer Mix to the purified samples followed by PCR amplification (10 cycles). Amplified libraries were purified using QIAquick MinElute columns (Qiagen) followed by size selection of 300bp using E-Gel SizeSelect Agarose Gels 2% (Thermo Fisher Scientific). Size selected libraries were controlled for correct size selection using the BioAnalyzer (Agilent) and the concentration was measured using the KAPA Library Quantification Kit (KAPA Biosystems).