Protocols

This website provides access to all standard protocols and procedures used at the PennVet Center for Host-Microbial Interactions (CHMI). If you’re affiliated with CHMI or a member of the Beiting Lab, then you can collaboratively edit any of these protocols. This wiki-style approach gives us a central place to develop our lab protocols, while always keeping the most up-to-date protocol in one convenient and publicly accessible place. If you’re not part of CHMI, no worries, you can still peruse the site to see our protocols. Comments and feedback are welcome!

Quantification and Quality Control

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Protocols for Quantification and Qualification of DNA, RNA, and NGS librariesTapestation ProtocolQubit ProtocolPicoGreen Protocol

Bulk RNA-seq and ATAC-seq

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Information and Protocols for all Bulk RNAseq and ATACseq library preparations and analysis currently used in the Beiting Lab📌RNA-seq FAQSIllumina Stranded mRNA-seqIllumina Stranded Total RNA-seqTakara Low Input RNA-seqTakara Low Input Degraded Total RNA-seq (Pico v3/FFPE-seq)Illumina miRNA-seqIllumina ATAC-seq Library PrepSorting cells for RNA-seq💻Bioinformatics for RNAseq/ATACseq

Single-Cell RNA-seq

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The Beiting Lab exclusively uses 10X Genomics kits and Chromium controllers to generate single-cell RNA-seq data. See below for more information and protocols currently used in the lab.📌scRNA-seq FAQs📌10X Genomics FAQs🗓️Sign Up: 10X Chromium Controller and Deep Well Thermocycler

Considerations for low-plex pools

Index combinations for low-plex pools

Chromium Next GEM Single-Cell Official Protocols (recommended):

3’ Kit v3.1 Kit3’ Kit v3.1 Feature Barcoding KitFLEX KitATAC KitMultiome KitMucosal cell isolation for scRNA-seq10X Feature Barcoding CITE-Seq (TotalSeq B)

Internal Protocols:

10X CITE-seq and Cell Hashing RNAseq (TotalSeq A)10X RNAseq Library Prep10X ATACseq💻Bioinformatics for Single Cell 🧪Cell Digest for Tissue Samples stored in CryoStore (CS) 10

Spatial transcriptomics

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Information and Protocols for spatialIntestinal Swiss Rolls

Cell Culture

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Information and Protocols for Cell Culture🧫3D 'organotypic' culture🔬Microscopy for 3D cultures

Legacy Protocols

📜Legacy Protocols

Microbial Genomics

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Protocols and Information for Microbial Genomics experiments and analysis📌Microbiome FAQs16S Amplicon SeqIllumina DNA Prep (Nextera Flex)Hack Flex - ManualHack Flex - Zephyr🧫Colony DNA Extraction for Illumina DNA Prep or HackFlex16S Sanger Sequencing💻Bioinformatics for Microbial Genomics

Extractions

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DNA/RNA extraction protocols🧲ZymoBiomics™ 96 MagBead DNA Extraction

Linux Server

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Information for the CHMI Linux Servers👩🏽‍💻Using the CHMI Linux serverConnecting to our RStudio server💻Software we use💽Downloading from BaseSpace

Mobile Microbiology Lab

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Information for field-portable molecular biology tools🌊Field AMR SOPsBiomeme Portable qPCR - GeneralSetting up the MinION/MinIT💩BioPoo Assay on eDNA

Loading Sequencers

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Information for Sequencers within CHMI and available to the LabNextSeq 2000📌NextSeq 1000/2000 Library Kits (Full View)MiniSeqNovaSeq 6000Illumina Instrument Troubleshooting

Teaching

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Lab resources for learning how to analyze high-throughput sequencing data

DIYtranscriptomics: a full semester, virtual course on RNA-seq data analysis in R

☁️Metagenomics in the cloudTranscriptomics in the cloudConda for bioinformaticsUsing Github CopilotBash basicsGit basics