The largest database of trusted experimental protocols

Single read 250bp run

Manufactured by Illumina

The Single read 250bp run is a core function of Illumina's sequencing platforms. It provides sequencing read lengths up to 250 base pairs, which can be used for a variety of genomic applications.

Automatically generated - may contain errors

2 protocols using single read 250bp run

1

Single-Cell TCR Sequencing of T Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols
Naive CD4+ (CD4+CD25Foxp3 ), TripleloTreg (CD4+CD25loFoxp3+GITRlo PD-1lo) or TriplehiTreg (CD4+CD25hiFoxp3+GITRhiPD-1hi) cell populations were sorted from 3 replicate groups (2 mice per group) of single TCRβ-tg (B6.YAe62 β˜ tg+TCRα+/–) mice were sorted to 98% purity (FACS Aria, BD Biosciences). RNA was isolated using Trizol and precipitated with RNase free glycogen (Invitrogen) following the manufactures protocol. cDNA was prepared using oligo-dT’s (Promega) and Omniscript RT kit (Qiagen). cDNA was amplified with 20 rounds PCR with generic Vα2 primer (5’-CCCTGGGGAAGGCCCTGCTCTCCTGATA-3’) and TCR Cα primer (5’-GGTACACAGCAGGTTCTGGGTTCTGGATG-3’). 1/10th volume of the first round PCR was amplified with an additional 20 rounds of PCR using barcoded primers, for post sequence identification of originating T cell population, containing Illunima PE read primer and P5/7 regions, respectively. The resulting 300bp fragment was gel purified (Gene Clean II, MP Biomedicals) and sequenced on a MiSeq using a single read 250bp run (Illumina). Sequence data sets were parsed by barcode using the program fastq-multx59 and clonotypes for each population were tabulated using TCRklass60 (link).
+ Open protocol
+ Expand
2

Single-Cell TCR Sequencing of T Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols
Naive CD4+ (CD4+CD25Foxp3 ), TripleloTreg (CD4+CD25loFoxp3+GITRlo PD-1lo) or TriplehiTreg (CD4+CD25hiFoxp3+GITRhiPD-1hi) cell populations were sorted from 3 replicate groups (2 mice per group) of single TCRβ-tg (B6.YAe62 β˜ tg+TCRα+/–) mice were sorted to 98% purity (FACS Aria, BD Biosciences). RNA was isolated using Trizol and precipitated with RNase free glycogen (Invitrogen) following the manufactures protocol. cDNA was prepared using oligo-dT’s (Promega) and Omniscript RT kit (Qiagen). cDNA was amplified with 20 rounds PCR with generic Vα2 primer (5’-CCCTGGGGAAGGCCCTGCTCTCCTGATA-3’) and TCR Cα primer (5’-GGTACACAGCAGGTTCTGGGTTCTGGATG-3’). 1/10th volume of the first round PCR was amplified with an additional 20 rounds of PCR using barcoded primers, for post sequence identification of originating T cell population, containing Illunima PE read primer and P5/7 regions, respectively. The resulting 300bp fragment was gel purified (Gene Clean II, MP Biomedicals) and sequenced on a MiSeq using a single read 250bp run (Illumina). Sequence data sets were parsed by barcode using the program fastq-multx59 and clonotypes for each population were tabulated using TCRklass60 (link).
+ Open protocol
+ Expand

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?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!