Protein a and protein g dynabeads

Approximately 100 μl of D. eugracilis ovaries were dissected into PBS on ice. The tissue was homogenised on ice twice in 0.3 ml of the RIPA buffer. The lysate was cleared by centrifugation and diluted with 2.4 ml of IP dilution buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl) and split into 2 reactions. Around 5 μg of antibodies against Piwi and Aubergine were each coupled to 50:50 of protein-A and protein-G Dynabeads (Life Technologies). Lysates were incubated with bead-coupled antibodies, rotating at 4°C overnight. Subsequently, the beads were captured and washed 5 times with IP wash buffer (50 mM Tris–HCl (pH 7.5), 500 mM NaCl, 2 mM MgCl2, 10% glycerol, 1% Empigen). For Aub IP, 150 mM NaCl was used instead of 500 mM NaCl. The IP was eluted in 10mM DTT and 0.1% SDS in 1× TE at 85 degrees. The bound RNA was extracted using acid-phenol:chloroform followed by 2-propanol precipitation, mixed with the radio-labelled spikes before the size selection. The size-selected RNA was oxidised before ligating the adaptors.

Cells were fixed with 1% formaldehyde and subsequently quenched with glycine. After washing with PBS, the fixed samples were suspended in cell lysis buffer (5 mM PIPES-KCl pH 8.0, 85 mM KCl, 0.5% NP40), and collected nuclei were stored at −80 °C as nuclear pellets or nuclear lysates dissolved in nuclei lysis buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS). For MLX ChIP, lysates were thawed and sonicated with Sonifier (BRANSON) to obtain chromatin fragments of 300–1,000 bp, and 10-fold-diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH8.0, 1.2 mM EDTA, 0.01% SDS, 1.1% Triron100×, 167 mM NaCl). After double-dilution with a sonication buffer (90 mM Hepes pH 7.9, 220 mM NaCl, 10 mM EDTA, 1% NP 40, 0.2% sodium deoxycholate, 0.2% SDS), nuclei were homogenized with a Bioruptor (Tosho denki). After washing, a complex of nuclear proteins/DNAs and antibodies against MLX (Cell Signaling technology; D8G6W; 1:100 dilution) was retrieved with Protein A- and Protein G-Dynabeads (Life Technologies). After the cross-linking was reversed, chromatin fragments were treated with RNase A and proteinase K. DNA was purified with phenol-chloroform extraction or Ampure XP (Beckman Coulter). In the ChIP-qPCR analyses, the values from the immunoprecipitated samples were normalized to input DNA. Primer sequences are listed in Table S2, Supporting Information.

The experimental protocol was performed exactly as described previously [59 (link)]. Briefly, an oligonucleotide pool was synthesized that contains sequences coding for peptides of 31 amino acids that tile along the length of the Wuhan-Hu-1 Spike protein sequence [92 (link)] in 1 amino acid increments. For each peptide with the wildtype sequence, 19 variations were included that have a single mutation at the middle amino acid, resulting in a total library size of 24,820 unique sequences. The oligonucleotide pool was cloned into T7 phage, followed by amplification of the phage library; this step was performed twice independently to generate biological duplicate phage libraries. The phage library was incubated with a serum or plasma sample, then bound antibody-phage complexes were immunoprecipitated using Protein A and Protein G Dynabeads (Invitrogen). Bound phage were lysed, and DNA was amplified by PCR and cleaned prior to sequencing on an Illumina MiSeq or HiSeq 2500 with single end reads. Demultiplexing and read alignment were also performed as described previously [60 (link)].