Ultra 2 directional rna library prep kit

Single‐cell suspensions were prepared from spleens isolated from naive (n = 3 per group) or from LCMV‐WE‐infected (n = 4 per group) WT or Trail^−/− mice, 1 day postinfection. NK cells (defined as NK1.1⁺ and CD3^‐ cells) were sort‐purified by flow cytometry and resuspended in TRI‐reagent (Sigma‐Aldrich). RNA was isolated according to the manufacturer's instructions, and RNA concentration and integrity were assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA).
Barcoded stranded mRNA sequencing libraries were prepared from high‐quality total RNA samples (~10 ng/sample) using combination of the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490, Ipswich, MA, USA) to enrich the samples for polyadenylated RNA transcripts and the Ultra II Directional RNA Library Prep Kit (NEB #E7760). Obtained libraries that passed the quality check step were pooled in equimolar amounts, and 1.8 pM solution of this pool was loaded on the Illumina sequencer NextSeq 500 and sequenced uni‐directionally, generating ~500 million reads, each 85 bases long. Library preparation and sequencing was performed at the EMBL Genomics Core Facilities (GeneCore, Heidelberg, Germany).

RNA samples were extracted from mESCs (n = 3 biological replicates) using Trizol reagent (Invitrogen) and subjected to DNase digestion with Turbo DNase (Ambion AM2238). RNA samples were then rRNA depleted using FastSelect -rRNA HMR (Qiagen) and converted to cDNA using Ultra II Directional RNA Library Prep Kit (NEB E7760).

Nascent RNA-seq was performed as previously described (Kerry et al. 2017 (link)), after 96 h RUNX1 (SEM) or KMT2A-AFF1 (RS4;11) KD. Briefly, 1 × 10⁸ SEM cells were treated with 500 μM 4-thiouridine (4-SU) for 1 h. Cells were lysed with TRIzol (Invitrogen), and total RNA was precipitated and DNase I-treated. 4-SU-incorporated RNA was purified by biotinylation and streptavidin bead pulldown. DNA libraries were prepared using the Ultra II Directional RNA library prep kit (NEB, E7765) and sequenced by paired-end sequencing using a NextSeq 500 (Illumina).

Samples for RNA-Seq analysis included emasculated but unpollinated ovaries (E) as a control (absence of pollination, pollen tube growth and fertilization), and ovaries from IP6h, IP24h, DP24h, and XP24h style removal treatments. These samples were selected based on pollen tube growth observations; IP6h represented partial pollen tube growth (no penetration into the ovaries and hence no fertilization), IP24h represented full pollen penetration into the ovaries with fertilization, XP24h represented full pollen tube penetration without fertilization, while DP24h represented pollination only (no pollen tube growth and no fertilization). All samples were collected 48 h after pollination (2 DAA), and each sample contained 5 ovaries with three replications. Total RNA was extracted from the ovary samples using the RNeasy^® Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Treatment with DNase I (Nippon Gene, Tokyo, Japan) was carried out to remove genomic DNA contamination. mRNA was then purified from the total RNA using a poly(A) mRNA magnetic isolation module kit (New England BioLabs). The pure mRNA was used to construct paired-end libraries for Illumina using the Ultra™ II directional RNA library prep kit (New England BioLabs), and sequencing was performed on an Illumina NovaSeq 6000 platform (Illumina, Inc.).

CUT&RUN was performed as previously described (30 (link),31 (link)) using EZH2, H3K27me3 and IgG antibodies on 5 million cells per sample. DNA obtained from Drosophila melanogaster S2 cells was sonicated to 200bp and 1ng was added to each CUT&RUN sample as a heterologous spike-in control. ChIP was performed as previously described (32 (link)) using 9 μg of ATRX antibody (21 (link)) and 6 million cells per sample. For RNA-sequencing, RNA samples were extracted from Day 0 mESCs and Day 6 NPCs using Trizol reagent (Invitrogen) and subjected to DNase digestion with Turbo DNase (Ambion AM2238), then rRNA-depleted using FastSelect -rRNA HMR (Qiagen) and converted to cDNA using Ultra II Directional RNA Library Prep Kit (NEB E7760). DNA and cDNA samples were end-repaired using End-Repair Mix (Enzymatics), A-tailed using Klenow exonuclease minus (Enzymatics), purified with MinElute columns (Qiagen), and ligated to Illumina adapters (NEB #E7600) with T4 DNA ligase (Enzymatics). Size selection for fragments >150 bp was performed with AMpure XP (Beckman Coulter). Libraries were PCR amplified with barcoded adapters for Illumina sequencing (NEB #E7600) using Q5 DNA polymerase (NEB #M0491) and purified with MinElute. Sequencing was performed on a NextSeq 500 instrument (Illumina) with 38 × 2 paired-end cycles.

Starting with 1 µg RNA from total, light polysome (40S + 60S + 80S) and heavy polysome (tetrasome and heavier) fractions, we enriched mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S). NGS libraries were then prepared with NEBNext using Ultra II Directional RNA Library Prep Kit for library construction in a poly(A) mRNA enrichment workflow (E7760S) according to the manufacturer’s protocol. Individual libraries were purified with Ampure XP beads, analysed on an Agilent Bioanalyzer 2100 (HS DNA) for size and quality then quantified using Qubit 2.0. A first round (wild-type and uL30bb for light and heavy polysome fractions, untreated and staurosporine treated samples) of sequencing was performed on Illumina’s NextSeq 500 sequencer. Two pools (one for each N) of 8 libraries/pool were sequenced in 2 separate runs using NextSeq 500/550 High output v2 kit (150 cycles) FC-404-2002 achieving a mean depth of 70 million reads per sample. A subsequent round (Wild-type and ul30bb total extracts; ul30aa total light, and heavy polysome fractions) of sequencing was performed with a pool of 20 samples for a mean depth of 30 million reads per sample. Two biological replicates of WT, R28P-30A and R28P-30B were run similarly as a pool of 18 samples.