title: GSM5420703: ILM S1; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue samples were stored in RNAlater (Qiagen) at 2-8° until sequencing was performed. RNA extraction, library preparation and RNA sequencing were conducted at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Germany; www.kfb-regensburg.de) as previously described (Boeck et al., 2020). In brief, total RNA was extracted from RNV and ILM tissue samples and stabilized in RNAprotect Cell Reagent according to the RNeasy Plus Micro Kit protocol (Qiagen). After pelleting, the RNAprotect buffer was removed, replaced by RLT Plus buffer and the samples were homogenized by vortexing for 30 sec. Genomic DNA contamination was eliminated using gDNA Eliminator spin columns. Next, ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally, total RNA was eluted in 12 μl of nuclease-free water. RNA sequencing analysis was performed for 24 samples. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories) was used to generate first-strand cDNA from 750 pg total-RNA. Double-stranded cDNA was amplified by LD PCR (12 cycles) and purified via magnetic bead clean-up. Library preparation was carried out, as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). 150 pg of input cDNA was tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems). Equimolar amounts of each library were pooled, and the pools then used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in bcl files, which were converted into fastq files with the bcl2fastq v2.18 software.