Real time analysis v2

Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep for NeoPrep kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol. An input of 100 ng total RNA was used for each sample. Additional resuspension buffer was added to each library to obtain a final total volume of about 24 μL. The concentration of each library was determined using either the KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems) or the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). Libraries were normalized to 10 nM and combined into pools of 8–16 samples. Dilutions of libraries for quantitation and pooling was done using 10 mM Tris-HCl, pH 8.5.
Prior to sequencing at Michigan State University Research Technology Support Facility (MSU), the quality and quantity of library pools were determined by MSU using a combination of Qubit dsDNA HS, either Caliper LabChipGX HS DNA or Agilent Bioanalyzer High Sensitivity DNA and the Kapa Illumina Library Quantification qPCR assays. Each pool was loaded onto one lane of an Illumina HiSeq 4000 flow cell and sequencing performed in a 1 × 50bp single read format using HiSeq 4000 SBS reagents. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.

Total RNA was extracted from quadruplicates of whole rosettes grown in the DEPI system at midday of R1, R2, and R3 using the OMEGA E.Z.N.A Plant RNA kit (catalog no. R6827) according to the manufacturer's protocol. Respective mRNA libraries were prepared with an Illumina TruSeq Stranded mRNA Library Preparation Kit (Cat. No. 20020595) using a Perkin Elmer Sciclone G3 robot following manufacturer's recommendations. Completed libraries were assessed for quality and quantified using a combination of Qubit dsDNA HS and Advanced Analytical Fragment Analyzer High Sensitivity DNA NGS assays. cDNA Libraries were quantified using the Kapa Biosystems Illumina Library Quantification qPCR kit. Each pool was loaded onto one lane of an Illumina HiSeq 4000 flow cell and sequenced in a 1 × 50bp single read format using HiSeq SBS reagents. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7, and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.

Sequencing of the 10x Genomics Single Cell 3’ Gene Expression library was prepared from the cells dissociated from four pooled organoids per condition at day 15 of hHO differentiation. The libraries were prepared using the 10x Chromium Next GEM Single Cell 3’ Kit, v3.1 and associated components. Completed libraries were QC’d and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Invitrogen Collibri Library Quantification qPCR assays. The library was loaded onto an Illumina NextSeq 500 v1.5 Mid Output flow cell. Sequencing was performed in a custom paired end format: 28 bases for read 1 which captures the 10x cell barcode and Unique Molecular Identifier (UMI), and 90 bases for read 2 which is the RNA portion of the library fragment. Base calling was done by Illumina Real Time Analysis (RTA) v2.4.11 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0. After demultiplexing and FastQ conversion, secondary analysis was performed using cellranger count (v6.0.0). Analysis was executed using 10X Loupe Browser 6 for k-means clustering and t-SNE visualization, Enrichr (https://maayanlab.cloud/Enrichr/) for gene ontologies and DiVenn 2.0 (https://divenn.tch.harvard.edu/) for differentially expressed genes analysis.