Standard Operating Procedures Used in Sample Processing

GeneLab Sequencing group uses standard procedures for sample processing and sequencing. These Standard Operating Procedures (SOPs) are available on the GeneLab Sample Processing Github page - https://github.com/nasa/GeneLab-sampleProcessing

Tissue Storage and Cutting

  • 1.2 Frozen tissue cutting

    This SOP describes the steps required to safely section a tissue prior the extraction of nucleic acids. The procedure, if followed correctly will allow portioning a piece of tissue without thawing and compromising the original biological sample.

Homogenization

  • 2.1 Tissue homogenization using Bullet Blender Gold Bead Beater

    This SOP describes the steps required to lyse and homogenize biological material using a Bullet Blender 24 Gold bead beater. This procedure was validated for downstream RNA/DNA extraction using the Qiagen AllPrep kits (SOP#3.1), it is also possible to use the procedure with downstream RNA extraction using Trizol (SOP#3.2).

  • 2.2 Tissue homogenization using Polytron Rotor Stator Homogenizer

    This SOP describes the steps required to homogenize biological samples using the handheld rotor stator homogenizer Polytron. This type of homogenator allow for a larger lysis buffer volume and is used mainly for samples that require larger yield and/or not yet optimized for bead homogenization. This procedure is currently routinely used for mouse skin RNA extraction that requires a downstream Trizol extraction (SOP#3.2).

Extraction

  • 3.1 QIAGEN AllPrep DNA/RNA Mini (Cat#80204) with QIAGEN RNase-Free DNase Set. (Cat#79254)

    This SOP describes the steps required to extract both RNA and DNA from mammalian tissues. The extraction kit used in this procedure is Qiagen AllPrep DNA/RNA Mini. In addition, in this procedure we describe steps for depleting the isolated RNA from DNA using QIAgen RNase-Free DNase set. This step is required for RNA that will be used for sequencing. It is strongly advised to read the AllPrep DNA/RNA Mini-Handbook in full.

  • 3.2 TRIzol RNA Extraction with QIAGEN RNase-Free DNase Set and QIAGEN Allprep

    This SOP describes the steps required to extract RNA from mammalian tissue using the TRIzol reagent for isolation and Qiagen Allprep mini kit for clean-up. In addition, in this procedure we describe steps for depleting the isolated RNA from DNA using QIAgen RNase-Free DNase set. This step is required for RNA that will be used for sequencing. It is strongly advised to read the AllPrep DNA/RNA Mini-Handbook in full.

DNA/RNA QC and Troubleshooting

  • 4.1 RNA/DNA/miRNA/cDNA quantification using Qubit Fluorimeter

    This SOP describes the steps required to perform quantification of RNA/DNA or cDNA using the Invitrogen (Thermo Fisher Scientific) fluorimeter – Qubit and the Qubit kits. Flourimetric methods are advantageous over spectrophotometric methods since they are more precise and specific to the molecule being measured.

  • 4.2 QC genomic DNA

    This SOP describes the steps required to perform automated electrophoresis of genomic DNA to assess DNA quality using the Agilent 4200 TapeStation System and Agilent Genomic DNA TapeStation reagents. Any number of samples can be analyzed between 1 and 96.

  • 4.3 Quality analysis of RNA using Agilent Bioanalyzer 2100 System

    This SOP describes the steps required to perform automated electrophoresis of RNA samples. This procedure is using the Agilent 2100 Bioanalyzer System and Agilent RNA 6000 Nano and Pico kits. This procedure will generate a gel image of the RNA as well as RIN and DV200 values, those are recorded and used to track sample quality.

Library Preparation

Library QC and Troubleshooting

  • 6.3 QC cDNA

    This SOP lists the steps for DNA quantification of sequencing libraries using an Agilent D1000 TapeStation.

Sequencing Parameters

Sequencing QC and Troubleshooting

If you have any questions, please contact us at ARC-DL-GeneLab-sequencing-group@mail.nasa.gov.