RNA-Seq Library Preparation from Sorted Cells

Cells were sorted as described in “Tissue Preparation and Flow Cytometry” and immediately lysed with RLT Buffer from the Qiagen RNeasy Plus Mini kit or RNA extraction buffer from PicoPure RNA isolation kit (Arcturus Bioscience, Inc.). Cell lysates were stored at −80°C until RNA was extracted. RNA isolations were performed using the Qiagen RNeasy Plus Mini kit or PicoPure RNA isolation kit. RNA quality and quantity were measured using the Agilent Technologies High Sensitivity RNA ScreenTape System. SMART-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories, Inc.) was used for full-length cDNA synthesis, and the Nextera XT DNA sample preparation kit (Illumina Inc.) was used for library preparation. DNA libraries were sequenced on an Illumina NextSeq 500 instrument with a target read depth of ∼10 million aligned reads per sample. The pool was denatured and diluted, resulting in a 2.5 pM DNA solution. PhiX control was spiked at 1%, and the pool was sequenced by 1 × 75 cycles using the NextSeq 500 High Output reagent kit (Illumina Inc.).

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Tsai F., Homan P.J., Agrawal H., Misharin A.V., Abdala-Valencia H., Haines GK I.I.I., Dominguez S., Bloomfield C.L., Saber R., Chang A., Mohan C., Hutcheson J., Davidson A., Budinger G.R., Bouillet P., Dorfleutner A., Stehlik C., Winter D.R., Cuda C.M, & Perlman H. (2017). Bim suppresses the development of SLE by limiting myeloid inflammatory responses. The Journal of Experimental Medicine, 214(12), 3753-3773.

Publication 2017

RNeasy Plus Mini kit PicoPure RNA isolation kit High Sensitivity RNA ScreenTape System SMART-Seq v4 Ultra Low Input RNA kit Nextera XT DNA sample preparation kit NextSeq 500 instrument NextSeq 500 High Output reagent kit

Buffer Cdna Cell Dna libraries Flow cytometry Isolation Sensitivity Synthesis Tissue

Corresponding Organization : Northwestern University

Other organizations : Icahn School of Medicine at Mount Sinai, University of Chicago, University of Houston, Feinstein Institute for Medical Research, Walter and Eliza Hall Institute of Medical Research

Top 5 similar protocols

Variable analysis

independent variables

Cell sorting method (described in 'Tissue Preparation and Flow Cytometry')

dependent variables

RNA quality and quantity
Gene expression (as measured by RNA sequencing)

control variables

Lysis buffer (RLT Buffer from Qiagen RNeasy Plus Mini kit or RNA extraction buffer from PicoPure RNA isolation kit)
RNA extraction method (Qiagen RNeasy Plus Mini kit or PicoPure RNA isolation kit)
CDNA synthesis method (SMART-Seq v4 Ultra Low Input RNA kit)
Library preparation method (Nextera XT DNA sample preparation kit)
Sequencing platform (Illumina NextSeq 500)
Sequencing depth (target of ~10 million aligned reads per sample)
Sequencing parameters (1 × 75 cycles, NextSeq 500 High Output reagent kit, 2.5 pM DNA solution, 1% PhiX control spike-in)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

Based on most similar protocols

Use the Qiagen RNeasy Plus Mini kit or PicoPure RNA isolation kit to extract RNA from cell lysates (Input protocol, Protocol 1, Protocol 2, Protocol 4, Protocol 5).

Perform an additional on-column DNase treatment during RNA extraction using the RNase-Free DNase Set (Qiagen) to remove genomic DNA contamination (Protocol 1, Protocol 2, Protocol 5).

Measure RNA quality and quantity using the Agilent Technologies High Sensitivity RNA ScreenTape System (Input protocol, Protocol 1, Protocol 2, Protocol 4).

Use the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories, Inc.) for full-length cDNA synthesis (Input protocol, Protocol 1, Protocol 5).

Use the Nextera XT DNA sample preparation kit (Illumina Inc.) for library preparation (Input protocol, Protocol 1, Protocol 3).

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!