GSM4972536: TPO4 9-cis RA rep 1 RNA; Homo sapiens; RNA-Seq
RNA-seq: Pellets were snap frozen and stored at ‐80 ⁰C. RNA from snap frozen pellets of drug treated organoids was extracted using the RNeasy RNA extraction kit (Qiagen) with DnaseI treatment. ATAC-seq: Cells were pelleted and resuspended in Recovery Cell Culture Freezing Medium (GIBCO) at -80°C to maintain native chromatin. At the start of the experiment, cells were thawed quickly at 37°C and washed twice with PBS. RNA-seq: A total amount of 1 µg RNA per sample was used for the preparation of RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (KAPA Biosystems). In short, oligo hybridization and rRNA depletion, rRNA depletion cleanup, Dnase digestion, Dnase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94 ⁰C for 6:30 min. First strand synthesis, second strand synthesis, and A‐ tailing was performed according to protocol. For the adapter ligation, a 7 μM stock was used (NextFlex DNA barcodes, Bioo Scientific). First, and second post‐ligation cleanup was performed according protocol. For the library amplification, 6 cycli were used. The library amplification cleanup was performed using a 0.8x bead‐based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer (Agilent Technologies), library concentration was measured using the DeNovix dsDNA High Sensitivity Assay (DeNovix). Sequencing was performed using an Illumina NextSeq 500, 50‐bp paired‐end reads were generated. ATAC-seq: Nuclei isolated in hypotonic buffer were tagmented for 5 minutes with in-house generated Tn5 while shaking at 37°C and DNA was purified using 2x SPRI beads (Ampure) purification. DNA was subsequently PCR amplified by using KAPA HiFi Hotstart Ready Mix (KAPA Biosystems) and Nextera Index Kit (Illumina) primers followed by reverse phase 0.65 x SPRI beads purification and a QIAquick Spin Column (QIAGEN) purification. Amplified DNA libraries were sequenced with an Illumina NextSeq 500 at a read length of 38 bp.