Nextseq high output kit v2 reagents

ATAC‐seq libraries were prepared using 50,000 cells, as previously described (Buenrostro et al., 2015 ), with the following modifications: Digitonin was added to the transposition reaction at a final concentration of 0.01%; the transposition reaction was purified using the Genomic DNA Clean & Concentrator‐10 kit (Zymo Research Corporation); PCR amplification was carried out using the Nextera DNA Library Prep (Illumina) Index Adapters, Nextera PCR Master Mix, and PCR Primer Cocktail for 10 cycles of PCR; PCR reaction was purified using 1.7× SPRI beads (Agencourt AMPure XP, Beckman Coulter). Libraries were checked for quality and concentration using the DNA High‐Sensitivity LabChip assay (Agilent Technologies) and quantitative PCR (KAPA Biosystems), according to the manufacturer's instructions. Libraries were pooled and sequenced 75 bp paired‐end on the NextSeq 500 (Illumina) using NextSeq High Output Kit v2 reagents (Illumina).

Tissues preserved in RNAlater were homogenized in TRIzol (ThermoFisher Scientific) using a gentleMACS dissociator (Miltenyi Biotec Inc). Total RNA was isolated using the miRNeasy Mini kit (Qiagen) according to manufacturer’s protocols, including the optional DNase digest step. RNA quality and concentration were assessed using the RNA 6000 Nano LabChip assay on the 2100 Bioanalyzer instrument and Nanodrop 2000 spectrophotometer (Thermo Scientific). Prior to 2016, non-stranded libraries were constructed using TruSeq RNA Library Prep Kit v2 (Illumina). Stranded libraries were prepared using the KAPA mRNA HyperPrep Kit (KAPA Biosystems), according to the manufacturer’s instructions. Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. Libraries were checked for quality and concentration using the DNA 1000 assay (Agilent Technologies) and quantitative PCR (KAPA Biosystems), according to the manufacturers’ instructions. Libraries were pooled and sequenced 75 bp paired-end on the NextSeq 500 (Illumina) using NextSeq High Output Kit v2 reagents (Illumina), or 100 bp paired-end on the HiSeq2500 (Illumina) using TruSeq SBS v3 reagents (Illumina).