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Tag: gBlock
cfDNA Standards For Molecular Testing
Multiplexed ctDNA fragments (~150bp) mixed with nucleosomally fragmented wildtype cfDNA background in human plasma. The cell-derived ctDNA fragments are generated by Anchor’s unique multiplexed gene-editing method and are nucleosomally fragmented to around 150bp. The cell-derived variants are suitable for both the amplicon-based and capture-based methods. The synthetic ctDNA fragments are…
Extraction-free LAMP assays for generic detection of Old World Orthopoxviruses and specific detection of Mpox virus
Phylogenomic analysis of Orthopoxvirus genomes A phylogenomic analysis of 200 Orthopoxvirus genomes, identified 10 distinct clades within this genus, which are here named as phylogroups 1 to 10 and are denoted as OPV-PG-01 to OPV-PG-10 (Fig. 1) based on their nesting patterns (Supplementary Fig. S1), where the 100 MPV isolates included…
Unzipped chromosome-level genomes reveal allopolyploid nematode origin pattern as unreduced gamete hybridization
Nematode materials and species identification Mi, Mj, and Mg were collected from farmlands in Wuhan city of Hubei province, Longyan city of Fujian province, and Changsha city of Hunan Provinces, respectively. Two Ma samples were collected from farmlands in Shenyang city of Liaoning province and Shiping city of Yunnan province….
Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening
Construct design To transcribe and translate sequenced DNA clusters on an Illumina flow cell, our DNA constructs contained the following elements: a P5 adaptor, followed by a 28 nt unique barcode, a 27 nt unstructured spacer (5p UNS v2), a ribosome binding site, start codon, protein coding region, TolAk short linker, 2×…
Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity
Mice All animal use was approved by the Department of Comparative Medicine at the Massachusetts Institute of Technology (MIT) and the Institutional Animal Care and Use Committee under protocol no. 0714-076-17. Mice were housed with a 12-h light/12-h dark cycle with temperatures in the range 20–22 °C and 30–70% humidity. KrasLSL-G12D…
A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon
Cell lines and culture A549-hACE2 [a gift from Dr. Ben tenOever3], 293 T, HEK293, A549, and 3T3-L1 pre-adipocyte cell lines (refer to Supplementary Table 1) were cultured in DMEM and 10% FBS. U937, Calu-3, and HCT116 cells were cultured in RPMI, EMEM, and McCoy’s 5 A media, respectively, with 10% FBS. Caco-2 cells…
Hybrids of RNA viruses and viroid-like elements replicate in fungi
Ribozyme search of the Sequence Read Archive Observing that ribozymes are sufficiently short to be captured on a short sequence read (less than 100 nt), we reasoned it will be possible to screen large volumes of sequencing data to identify libraries potentially containing ribozyme agents. To this end we adapted…
Spontaneous tumor regression mediated by human T cells in a humanized immune system mouse model
Human CD34+ cell isolation Human fetal liver (FL) samples were obtained from Advanced Biosciences Resources (Alameda, CA) with proper consent. FL samples were cut in small fragments, treated for 25 min at 37 °C with Collagenase D (100 ng/mL; Roche). The cell suspension was prepared, and the human CD34+ cells were separated by…
Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods. DMS-MaP is a sequencing-based method that allows us to get a snapshot of RNA structure, and learn how it changes under different…
BdLT-Seq as a barcode decay-based method to unravel lineage-linked transcriptome plasticity
Cell lines Immortalised HA1E (hTERT and SV40ER) and tumourigenic HA1ER cells (hTERT, SV40ER and HRAS-G12V) from stepwise tumourigenesis models generated from normal human embryonic kidney cells were obtained from Dr. Hahn (Broad Institute of MIT and Harvard, Cambridge, USA). Cells were cultured in DMEM (1 g/L glucose) (Gibco, cat. #10567014) supplemented…
Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis
Cell culture IMR90 (CCL-186) and WI38 (AG06814-N) fibroblasts were purchased from ATCC and the Coriell Institute for Medical Research, respectively. IMR90 and WI38 fibroblasts were grown under 7.5% CO2 and 3% O2 in GlutaMax-DMEM (Gibco, 10569-010) supplemented with 0.1 mM non-essential amino acids (Corning, 25-025-Cl) and 15% fetal bovine serum (VWR/Seradigm,…
Trem2 H157Y increases soluble TREM2 production and reduces amyloid pathology | Molecular Neurodegeneration
Generation, genotyping, and off-target analysis of Trem2 H157Y knock-in mice Trem2 H157Y knock-in mice were generated via CRISPR/Cas9 by the Hope Center Transgenic Vectors Core of the Washington University [25]. CRISPR gRNAs for in vitro testing were identified using CRISPOR (crispor.tefor.net/) and synthesized as gBlocks (Integrated DNA Technologies, IDT) with…