4200 tapestation and high sensitivity d1k screen tape

For RNA-seq, between 62,000–210,000 ISCs were sorted directly into Buffer RLT and RNA was isolated using the RNeasy Mikro Kit (Qiagen). QC of samples was done to determine RNA quantity and quality prior to the processing by low input RNA-seq method. The concentration of RNA samples was measured using DS-11 spectrophotometer (DeNovix) and the integrity of RNA was determined by 2100 Bioanalyzer (Agilent Technologies). Approximately few ng of total RNA was used as an input material for the library generation using SMART-seq v4 Ultra Low Input RNA kit (Clontech). Size of the libraries was confirmed using 4200 TapeStation and High Sensitivity D1K screen tape (Agilent Technologies) and their concentration was determined by qPCR based method using Library quantification kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq4000 (Illumina) to generate >30M of single end 50 base pair reads.

Total RNA was isolated using the RNeasy kit (Qiagen) per manufacturer’s suggestions. Quality control of total RNA was done to determine sample quantity and quality. The concentration of RNA samples was determined using NanoDrop 8000 (Thermo Fisher Scientific), and the integrity of RNA was determined by Fragment Analyzer (Advanced Analytical Technologies). A total of 0.1 μg of RNA was used as input material for library preparation using TruSeq Stranded Total RNA Library Prep Kit (Illumina). Size of the libraries was confirmed using 4200 TapeStation and High Sensitivity D1K screen tape (Agilent Technologies), and concentration was determined by quantitative PCR–based method using library quantification kit (KAPA). Libraries were multiplexed and sequenced on Illumina HiSeq4000 (Illumina) to generate 30 million single-end 50–base pair reads.