Nebnext ultra 2 fs kit

TR14 scCircle-seq data were deposited in the European Genome-phenome Archive (EGA) under the accession number: EGAS00001007026. A detailed description of the single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq) protocol is available on Nature protocol exchange (DOI: 10.21203/rs.3.pex-2180/v1). In short, single cells were separated into individual wells of a 96-well plates using FACS. Separation of genomic DNA and mRNA was performed as described in the G&T-seq protocol by Macaulay et al. 2015^{49 (link)}. Genomic DNA of single cells was purified using 0.8× AMPure XP beads and subjected to exonuclease digestion and rolling-circle amplification as described in Chamorro González et al, 2023 (in press). All single-cell libraries were prepared using the NEBNext Ultra II FS kit (New England Biolabs) following the manufacturer’s instructions but using one-fourth volumes. Unique dual index primer pairs (New England Biolabs) were used to barcode single-cell libraries. Pooled libraries were sequenced on the Illumina HiSeq 4000 or the NovaSeq 6000 platform with 2 × 150bp paired-end reads for genomic DNA and circular DNA libraries and 2 × 75 bp paired-end reads for cDNA libraries.

The detailed ChIP protocol is in the Supplemental Material. For sequencing, DNA fragments were made into libraries using the NEBNext Ultra II FS kit (New England Biolabs). Libraries were pooled and sequenced on a NextSeq 500 platform (Illumina) with paired-end 150-nt runs at high output (developing seeds) or single-end 75-nt runs at high output (mature embryos). Adaptor sequences were removed using Trimmomatic (Bolger et al. 2014 (link)); trimmed reads were then aligned on the Arabidopsis TAIR10 genome using Bowtie2 (Langmead and Salzberg 2012 (link)). Duplicated reads are removed using the Picard Tool suite, and coverage was calculated and mapped to DMRs using Bedtools. All distributions were compared with the first WT sample distribution using the Kolmogorov–Smirnov test with R.