RNA sequencing from mouse, pig and human precision cut lung slices (PCLS)

Precision-cut lung slices (PCLS) have received increased attention as a relevant model for studying and modeling lung diseases. However, isolation of high quality RNA for performing non-biased genetic analysis, such as next generation sequencing (NGS) is challenging. Here we present a technique to isolate high quality RNA from limited amounts of tissue which is sufficient for running RNA sequencing and integration with existing computational pipelines and workflows. We show that this isolation technique is suitable for isolating RNA from mouse, pig and human PCLS, as well as PCLS derived from the lungs of patients with underlying disease, such as idiopathic pulmonary fibrosis. We then predict cell diversity within PCLS by deconvoluting our bulk RNA sequencing data from human PCLS with recently reported single cell RNA sequencing from donor tissue or patients with IPF using BISQUE. We find that BISQUE predicts changes in cellular composition which represents the disease states from which the PCLS were derived. For the library preparation, the TruSeq Stranded mRNA Library Prep Kit (Illumina) was used with an input of 150 ng RNA (determined by the Qubit RNA HS Kit), unless otherwise stated, following the manufacture's instruction with the exception that 13 cycles instead of 15 were used to enrich DNA fragments. The IDT for Illumina TruSeq RNA UD Indexes (96 indexes) was used to index the different samples for multiplexing. A King Fisher FLEX (Thermo Scientific) was used for automatization of the clean-up steps and a Mastercycler X50s (Eppendorf) was used for the incubations and PCR. The QuantIT dsDNA HS Assay Kit (Invitrogen) was used to determine the DNA concentration of the library. The size and quality of the library was tested using the LabChip GX Touch (Perkin Elmer) with the DNA 1K / 12K / Hi Sensitivity Assay LabChip (Perkin Elmer) and the LabChip DNA High Sensitivity Reagent Kit. A library input of 1.4 pM (with 1 % PhiX Control (Illumina)) was sequenced using the NextSeq 500 instrument (Illumina) with the NextSeq 500/550 High Output v2.5 Kit (Illumina). The FASTQ files created were de-multiplexed using the bclfastq2 software (Illumina) and the software FastQC (Babraham Bioinformatics) was used for the quality control of the raw data. The HISAT2 software was used for alignment of the reads to the reference genomes. All reference genomes were from the Ensembl database (Mouse GRCm38, Pig Ssacrofa11.1, and Human GRCh38) with the GTF annotation (release 99). For the assembly of the alignments into full transcripts and quantification of the expression levels of each gene/transcript, the StringTie software was used.