Fastselect rrna hmr

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).

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.

mNGS libraries were prepared from isolated pathogen RNA and converted to cDNA Illumina libraries using the NEBNext Ultra II RNA Library Prep Kit (New England BioLabs). Human rRNA was depleted via FastSelect -rRNA HMR (Qiagen). ERCC Spike-In Controls (ThermoFisher) were used to indicate potential library preparation errors and to calculate input RNA mass. The initial samples (n = 208) were sequenced on a NovaSeq6000 (Illumina) instrument as part of a pilot wet laboratory training at the Chan Zuckerberg BioHub in San Francisco, CA, and then the remainder of the study (n = 279) was performed on an iSeq100 (Illumina) in Phnom Penh, Cambodia, using 150-nucleotide paired-end sequencing. Water controls were included in each library preparation.

For each collected sample, nucleic acid was extracted using the quick-DNA/RNA Pathogen MagBead kit (Zymo Research). Extracted nucleic acid was treated with DNAse to isolate RNA alone and run on a TapeStation for quality control to examine RNA integrity. Water controls were used to characterize background contamination, as well as 25 pg of a positive control spike-in (RNA standard dilution series from External RNA Controls Consortium (ERCC)). Plate maps were designed to detect and minimize cross-contamination between wells by interspersing samples and water controls. Samples were run in two experimental batches (see S1 Table). FastSelect -rRNA HMR (Qiagen) was used for human RNA ribosomal depletion, which is known to knockdown ~98% of human ribosomal RNA targeting the cytoplasmic (5S,5.8S,18S,28S) and mitochondrial (12S,16S) rRNAs, and specifically targets human, mouse, rat, and other mammalian species [22 ]. RNA was reverse-transcribed to attain cDNA, which was used to construct and barcode sequencing libraries using the NEBNext Ultra II Library Prep Kit (New England Biolabs). The RNA sequencing libraries underwent 150-nt paired-end Illumina sequencing. The target reads were at least 5 million reads per sample to attain enough coverage depth per respective sample.