GSM7719856: 4-Ablation-p63-P2+15-d14-220726; Mus musculus; RNA-Seq
Organoid harvest was carried out using Trevigen Cultrex 3-D Cell Harvesting Kit. Organoids were stored in 1x RNA Shield at -80°C until RNA isolation (maximum storage time: 3 weeks). RNA of all samples was isolated in one run using ZYMO Quick-DNA/RNA Microprep Plus Kit. Library generation, quality control, and quantification:  150ng of total RNA per sample were utilized as input for mRNA enrichment procedure with 'NEBNext® Poly(A) mRNA Magnetic Isolation Module' (E7490L; New England Biolabs) followed by stranded cDNA library generation using 'NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina' (E7760L; New England Biolabs). All steps were performed as recommended in user manualE7760 (Version 1.0_02-2017; NEB) except that all reactions were downscaled to 2/3 of initial volumes.  cDNA libraries were barcoded by dual indexing approach, using 'NEBNext Multiplex Oligos for Illumina – 96 Unique Dual Index Primer Pairs' (6440S; New England Biolabs). All generated cDNA libraries were amplified with 10 cycles of final pcr.  One additional purification step was introduced at the end of the standard procedure, using 1.2x 'Agencourt® AMPure® XP Beads' (#A63881; Beckman Coulter, Inc.). Fragment length distribution of individual libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific).   Library denaturation and Sequencing run:  Equal molar amounts of individually barcoded libraries were pooled for a common sequencing run in which each analyzed library constituted 16.7% of overall flowcell / run capacity. The library pool was denatured with NaOH and was finally diluted to 1.8pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3ml of the denatured pool was loaded on an Illumina NextSeq 550 sequencer using a Mid Output Flowcell (130M cluster) for 2x75bp paired-end reads (20024904; Illumina). Sequencing was performed with the following settings: Sequence reads 1 and 2 with 76 bases each; Index reads 1 and 2 with 8 bases each.