Hiseq2500 150 bp paired end platform

Approximately 5,000 Lgr5+ HC progenitors and 5,000 Lgr5- SCs were isolated by FACS and split into three fractions for separate replicates. RNA-Seq libraries of FACS-purified cells were generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and the Illumina mRNA-Seq Sample Prep Kit. FACS-purified cells were suspended in 10 × lysis buffer. First strand and second strand cDNA synthesis, adaptor ligation, and PCR amplification were performed using the Illumina mRNA-Seq Sample Prep Kit. SPRI beads (Ampure XP, Beckman) were used in each purification step after RNA fragmentation for size selection. All libraries were analyzed for quality and concentration using an Agilent Bioanalyzer. Sequencing was performed using the Illumina HiSeq2500 150-bp Paired-End Platform, and FASTQ files of paired-end read files were generated.

Approximately 5000 GFP+ SCs were isolated by FACS and split into three fractions for separate replicates. RNA-seq libraries of FACS-purified cells were generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and the Illumina mRNA-Seq Sample Prep Kit. FACS-purified cells were suspended in 10× lysis buffer. First-strand and second-strand cDNA synthesis, adaptor ligation, and PCR amplification were performed using the Illumina mRNA-Seq Sample Prep Kit. SPRI beads (Ampure XP, Beckman) were used in each purification step after RNA fragmentation for size selection. All libraries were analyzed for quality and concentration using an Agilent Bioanalyzer. Sequencing was performed using the Illumina HiSeq2500 150-bp Paired-End Platform, and FASTQ files of paired-end read files were generated.