Ovation ultralow library system

Cells were sorted by flow cytometry into PBS, 50% FCS, and RNA was extracted using the RNeasy Plus Micro kit (Qiagen). After assessment using a Bioanalyzer (Agilent), RNA was processed for RNA-seq using an Ovation RNA-seq System V2 (Nugen), fragmented using the Covaris M220 ultrasonicator and bar-coded using Ovation Ultralow Library Systems (Nugen). Samples were sequenced using an Illumina HiSeq 4000, by running a single-read 50-bp protocol (Cancer Research UK Cambridge Institute). Sequence data were trimmed to remove adaptors and sequences with a quality score below 30 using Trim Galore (version 0.50, Babraham Bioinformatics) and then aligned to the mouse genome (GRCm38) using STAR (version 2.6.0a), and differential expression was calculated using DESeq2 (version 1.18.1).⁶⁴ (link)

ChIP experiments were performed as previously described [51 (link)]. Chromatin was extracted from 7-day-old whole seedlings (150 seedlings). Anti-H2Aub (Cell Signaling Technology, 8240S) and anti-H3K27me3 (Diagenode, C15410069) antibodies were used for chromatin immunoprecipitation. For ChIP-seq, two immunoprecipitations from independent biological replicates were processed for next-generation sequencing library preparation. All libraries were prepared by the Ovation® Ultralow Library Systems (NuGEN) following the manufacturer’s instruction using 80% of a typical ChIP as starting material. After amplification for 16 PCR cycles, DNA of a size range between 200 and 500 bp was purified from an agarose gel. Amplification was confirmed by testing an aliquot of the library before and after amplification by qPCR. Sequencing was carried out as single-end 100-nucleotide reads on an Illumina HiSeq by the Max Planck Genome Centre in Cologne. For ChIP-qPCR, amplification was performed using Sensi FAST SYBR & Fluorescein kit (Bioline) and an iQ5 Biorad system. Samples were normalized to input DNA prepared from a reverse cross-linked aliquot of each chromatin preparation. qPCR data are shown as the means of two replicates from a representative experiment. Primers used for ChIP-qPCR are shown in Additional file 1: Table S2.

Cells were sorted by flow cytometry into PBS, 50% FCS, and RNA was extracted using the RNeasy Plus Micro kit (Qiagen). After assessment using a Bioanalyzer (Agilent), RNA was processed for RNA-seq using an Ovation RNA-seq System V2 (Nugen), fragmented using the Covaris M220 ultrasonicator and bar-coded using Ovation Ultralow Library Systems (Nugen). Samples were sequenced using an Illumina HiSeq 4000, by running a single-read 50-bp protocol (Cancer Research UK Cambridge Institute). Sequence data were trimmed to remove adaptors and sequences with a quality score below 30 using Trim Galore (version 0.50, Babraham Bioinformatics) and then aligned to the mouse genome (GRCm38) using STAR (version 2.6.0a), and differential expression was calculated using DESeq2 (version 1.18.1) ^{8 (link)}.

ChIP was performed as previously described (24–26 (link)) with minor modifications. The following antibodies were used: PU.1 (sc-352), IRF8 (sc-6058x), JUNB (sc-46x), CEBPA (sc-61x), RUNX1 (sc-8563x), STAT6 (sc-981x), p65 (sc-372), H4ac (millipore 06-866), H3K4me1 (ab8895), H3K27ac (ab4729) and RNAPII-pS2 (ab5095). Libraries were prepared by Ovation Ultralow Library Systems (Nugen) from two biological replicates according to the manufacturer’s instructions.