Kapa hifi hotstart real time library amp kit

Cells were sorted directly into Nuclei EZ Storage Buffer (Sigma, NUC-101) and stored at -80°C. ATAC-seq libraries were prepared following the Omni-ATAC protocol described by Corces et al.^{27 (link)} with the following modifications: Tagmented DNA was amplified using the KAPA HiFi HotStart Real-Time Library Amp Kit (Roche) with modified Nextera dual indexed primers as listed in Supplementary Table 7. Amplified libraries were purified using Ampure XP beads (Beckman Coulter) with double-sided size selection (1^st bead selection: 0.5x; 2^nd bead selection: 1.2x) according to manufacturer’s protocol. Quantified and validated libraries (~150-1000 bp) were subjected to pair-end sequencing on HiSeq 4000 sequencing system (Illumina), resulting in >30 millions single end reads per sample.

Sequencing libraries were generated with an in-house optimized protocol for total viral RNA sequencing using Nextera XT (Illumina). Viral RNA was reverse transcribed into cDNA using SuperScript IV (Invitrogen) and random pentadecamers. RNA was then digested using RNaseH (New England Biolabs, number M02976), denatured, and random pentadecamers were annealed. NEBNext Ultra II Q5 Master Mix was added and samples incubated for 10 min at 72°C to complete second strand-synthesis. Ampure XP beads were used for DNA purification and Nextera XT (Illumina) was used for tagmentation (fragmentation and adapter addition) according to the manufacturer’s protocol. Libraries were amplified with indexing primers for 12 cycles using the NEBNext Ultra II Q5 Master Mix, size selected using Ampure XP beads, and real-time amplified using the KAPA HiFi HotStart Real-Time Library Amp kit (Roche). Library QC was performed, libraries were pooled at equimolar ratios, and sequenced on an Illumina NextSeq 500.