Hiseq rapid v2 flowcell

Extraction of genomic DNA was performed using the DNeasy PowerSoil HTP Kit (QIAGEN, Germantown, MD) with bead beating taking place for 2 min in a Mini-Beadbeater-96 (BioSpec Products, Bartlesville, OK). Genomic DNA was shipped to the Functional Genomics Unit at the Roy J. Carver Biotechnology Center for library preparation. PCR was performed for 35 cycles with 16S rRNA V4 primers for total bacteria (Walters et al., 2016 (link)) and SJ Clostridial specific primers (Hu et al., 2014 (link)) using the 48 Access Array IFC (Fluidigm, San Francisco, CA). The final size-selected amplicon pools were submitted to the DNA Services laboratory at the DNA Services laboratory at the Roy J. Carver Center at the University of Illinois at Urbana-Champaign. The final pools were quantified using Qubit (Life Technologies, Grand Island, NY) and then further quantified by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA), then pooled evenly. The pool was loaded onto 1 lane of a 2-lane HiSeq Rapid V2 flowcell at a concentration of 10 pM for cluster formation on the cBOT and then sequenced on an Illumina HiSeq 2,500 with version 2 Rapid SBS sequencing reagents. The libraries were sequenced from both ends of the molecules to a total read length of 250 nt from each end.

For each genotype, 2 μg of RNA from each of the three biological replicates was used for cDNA library preparation. The cDNA libraries were prepared following the TruSeq RNA Sample Preparation v2 low sample (LS) protocol guide (Illumina Inc., San Diego, CA United States) as described in Kottapalli et al. (2016) (link) manually. One-third of the samples were prepared using TruSeq stranded mRNA on the Neoprep^™ following manufacturer’s instructions.
Each of the 10 nM cDNA libraries was diluted to 4 nM with hybridization buffer and multiplexed (four samples were pooled). Pooled cDNA libraries were denatured with NaOH and normalized to 10 nM concentration. A final concentration of 5.4 pM was loaded onto the MiSeq Reagent cartridge (MiSeq Reagent Kit v2 300 cycles, Illumina Inc., San Diego, CA United States). For the sequencing on the HiSeq 2,500 platform, we pooled equimolar concentrations of 24 samples into one single Rapid run of 2 lanes. A final concentration of 6.3pM was loaded onto the HiSeq2500 sequencer (HiSeq 2500, Illumina Inc., San Diego, CA, United States). Paired end sequencing with 150 bp paired-end reads was performed on a HiSeq Rapid v2 flow cell from Illumina as per manufacturer’s protocols.