RNA-seq from human colonocytes cultured for 1 hr (replicate 2)
Poly-adenylated mRNAs were isolated from thawed cell lysates using the Dynabeads mRNA Direct Kit (Ambion). RNA-seq libraries were prepared using a protocol modified from the NEBNext Ultradirectional (NEB) library preparation protocol to use Barcodes from BIOOScientific added by ligation. The individual libraries were quantified using the KAPA real-time PCR system, following the manufacturer's instructions and using a custom-made series of standards obtained from serial dilutions of the phi-X DNA (Illumina). The libraries were then pooled and sequenced on two lanes of the Illumina Next-seq 500 in the Luca/Pique laboratory using the high output kits for 75 cycles to obtain paired-end reads for an average of over 40 million total reads per sample.