Cells were stained with Propidium Iodide (Life Technologies) for dead cell exclusion and sorted with a FACSARIA II (BD Biosciences). After removal of debris and other none-cellular particles, doublets and multiplets were excluded on two consecutive gating steps (Forward-scatter height (FSC-H) versus forward-scatter area (FSC-A), side-scatter area (SSC-A) versus side-scatter width (SSC-W)). Dead cells were excluded based on Propidium Iodide uptake identified on the FSC-H versus PE-Cy5.5 profile, individual EGFP+/tdTomato− cells were sorted into different wells of 96-well PCR plates (USA Scientific) containing CellsDirect 2x Reaction mix (Invitrogen) supplemented with 0.05U of SUPERase-In RNase Inhibitor (Invitrogen). Flow rate was kept constant at 300 cells/sec (precision: single-cell, nozzle: 100 μm). 96-well plates were immediately sealed and stored at −80°C for subsequent RNA processing.
96 well pcr plates
Manufactured by USA Scientific
Sourced in United States
96-well PCR plates are a type of laboratory equipment used in polymerase chain reaction (PCR) experiments. They provide a standardized format with 96 individual wells for conducting multiple PCR reactions simultaneously. The plates are made of a durable, high-quality material and are designed to be compatible with common PCR thermal cyclers.
Automatically generated - may contain errors
Lab products found in correlation
Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Cellsdirect 2x reaction mix, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Sytox red, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
4 protocols using 96 well pcr plates
1
Single-cell FACS isolation for RNA-seq
2
Single-cell RNA-seq by FACS Sorting
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Sytox Red (ThermoFisher Scientific, Waltham, MA, United States) was added to cell suspensions to identify dead cells. Cells were sorted with a FACSARIA II (BD Biosciences). Debris and non-cellular particles were removed. Cell doublets and multiplets were removed using two consecutive gating steps: forward-scatter height (FSC-H) vs. forward-scatter area (FSC-A) and side-scatter area (SSC-A) vs. side–scatter width (SSC-W). Dead and compromised cells were identified by Sytox Red uptake and eliminated based on SSC-A vs. Sytox Red sorting. mGFP + /tdTomato-negative cells were collected into individual wells of 96-well PCR plates (USA Scientific, Ocala, FL, United States) pre-filled with CellsDirect 2 X Reaction mix (Invitrogen) and 0.05 U of SUPERase-In RNase Inhibitor (Invitrogen, Carlsbad, CA, United States). Flow rates were held at 300 cells/s (“precision” set to “single cell”; nozzle; 100 μm). Plates containing cells were sealed and stored at -80°C until RNA isolation.
3
Quantifying Viral DNA Copy Number
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Unfixed livers were homogenized and DNA was extracted according to the manufacturer’s instructions (Qiagen, cat. no. 56605223). Standards were made using serial dilution of the parental viral plasmid. Both standards and extracted DNA were then loaded onto 96-well PCR plates (USA Scientific, cat. no. 21034). qPCR was then performed according to protocol, detected by SYBR Green (Bio-Rad, cat. no.1725174). The vector copy number per diploid genome was calculated from equations obtained from the standards and their Ct values. The average of mice per group was used for comparisons.
4
Thermal Shift Assay of eIF4A
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