Minelute pcr purification kit

Following gene-specific PCR, a tagging PCR reaction was performed to append unique barcode sequences to the gene-specific products from each sample and to add adapters specific for sequencing on the Illumina GAIIx platform. Samples were fingerprinted for multiplex sequencing using one of 48 barcodes (Illumina). Purified products (10 ng) were tagged using the following conditions: 1 cycle of 94°C for 2 min, 10 cycles of (94°C for 30 sec; 56°C for 30 sec; 68°C for 1 min), 1 cycle of 68°C for 10 min, and 4°C hold. These PCR products were then pooled and purified using Qiagen MinElute PCR purification kit, and quantified using the KAPA Library Quant kit (KAPA Biosystems, Cape Town, South Africa) following the manufacturer's instructions. All samples were normalized to 8.6 nM and pools of 15 samples per lane were prepared. Flow cell preparation and data acquisition were completed using Illumina's recommended protocols. Paired-end sequencing runs (2×151) were performed using the Illumina GAIIx platform.

Subconfluent, HUVEC-FUCCI were lifted and sorted into early G1 and late G1 cell cycle states, then immediately collected for ATAC-sequencing analysis (n = 4 biological replicates). Library preparation was performed as previously described^{77 (link)}. Briefly, cells were pelleted by 13,000 rpm for 1.5 min in a tabletop centrifuge, cells were resuspended in 50 μL Transposase Reaction Mix (25 μL 2x TD Buffer, 2.5 μL Transposase Enzyme from Illumina Nextera Cat# FC121-1030, 22 μL nuclease-free water, 0.5 μL 0.1% digitonin) and incubated for 30 min at 37 °C, then DNA was purified using a QIAGEN MinElute PCR Purification Kit (Cat# 28004), amplified using KAPA HiFi HotStart ReadyMix PCR kit, and sequenced tagged primers according to the published protocol, then amplified DNA was purified using AMPure Beads (Beckman Coulter Cat# A63880). Sequencing was performed at the Yale Center for Genomic Analysis (Illumina HiSeq4000). Raw read data was quality controlled with FastQC v0.11.9 (Babraham Bioinformatics), filtered and trimmed with Trimmomatic v0.33^{78 (link)}, then peaks were called with MACS2 v2.2.7.1⁷⁹ , and differential peak analysis was performed with HOMER v4.11 (UCSD).