Total RNA was isolated from HEK293T cells using BIO TRI RNA reagent (Bio-Lab Chemicals) and mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to manufacturer's protocol. Poly(A)-purified RNAs were taken for library preparation using a derivation of MARS-seq (massively parallel RNA sequencing) as described (17 (link)). Briefly, 10 ng of poly(A)+ RNA was taken for the first reverse transcription reaction using Illumina barcoded RT1 primer. Resultant barcoded cDNA samples were subsequently pooled according to Ct values of a house-keeping gene (GAPDH) (Quality control 1). Pooled cDNA was treated with Exonuclease I (NEB) to remove excess primers followed by second strand synthesis. After that, in-vitro transcription was performed using T7 RNA Polymerase (NEB) to generate RNA, which was later fragmented and ligated to an adaptor consisting of RD2 using T4 RNA ligase I (NEB) followed by the second reverse transcription reaction. The library so formed was amplified using Kapa Hifi ready mix (Roche). The amplified RNA libraries were sequenced using a high-throughput 75 bp kit (Illumina FC404-2005) on NEXTseq 500 sequencer.
Fc404 2005
Manufactured by Illumina
The FC404-2005 is a laboratory instrument designed for performing thermal cycling reactions, such as polymerase chain reaction (PCR) and other DNA amplification techniques. It provides precise temperature control and monitoring for efficient and reliable nucleic acid amplification. The core function of this product is to facilitate the thermal cycling process required for various molecular biology applications.
Automatically generated - may contain errors
2 protocols using fc404 2005
1
Poly(A)+ RNA Sequencing in HEK293T Cells
2
Automated RNA-seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the lysates used for TI-seq and Ribo-seq using Bio Tri RNA reagent. Total RNA was then cleaned up using Oligo d(T)25 Magnetic Beads (NEB S1419S) to isolate mRNA. RNA-seq libraries were prepared using a derivation of MARS-seq as described (47 (link)) to produce expression libraries with two replicates of each treatment. Approximately 25 ng RNA was taken for the first reverse transcription reaction using Illumina bar-coded RT1 primer. Resultant bar-coded cDNA samples were subsequently pooled according to cycle threshold values of the housekeeping gene (GAPDH) (Quality control 1). Pooled cDNA was treated with Exonuclease I to remove excess primers, followed by second-strand synthesis using NEB SSS module enzyme mix. After that, in vitro transcription was performed using NEB T7 RNA Pol mix to generate RNA, which was fragmented and ligated to an adaptor consisting of RD2 using T4 RNA ligase I (NEB), followed by a second reverse transcription reaction. The library was amplified using Kappa Hifi ready mix. The RNA libraries were sequenced using a high-throughput 75-bp kit (Illumina FC404-2005) on NEXTsEq. 500 sequencer.
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
Revolutionizing how scientists
search and build protocols!