METHOD FOR CREATING A cDNA SEQUENCING LIBRARY
A technology for sequencing libraries and sequences, applied in the field of creating cDNA libraries, can solve problems such as hindering detection and sacrificing mRNA sequencing coverage
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Embodiment 1
[0102] Fixed intracellular reverse transcription (RT) of RNA in isolated wells using barcoded random decamers (as primers) and biotinylated dNTPs ( figure 1 , 2 ). By using barcodes in RT reactions, it is possible to spatially encrypt different wells in the plate or in different tissue regions. Importantly, rRNA naturally occurring in structured form or in ribonucleoprotein complexes is not involved in RT and does not need to be excluded in subsequent steps. Biotinylated dNTPs are incorporated into the newly synthesized cDNA strands for subsequent purification steps using streptavidin-coated magnetic beads. After RT, cells were washed to remove excess RT primer. cDNA was retrieved from RNA / cDNA heteroduplexes using RNAseH ribonuclease, and supernatants from all wells were pooled into a single, larger single volume. It is important that fixed cells are not exposed to any lysis chemicals during this protocol. Preservation of protein epitopes allows the user to perform in do...
Embodiment 2
[0104] The RIF-Seq protocol is very scalable and can be used for single reactions, 96 / 384 / 1536-well plates, or tissue samples. RIF-Seq can also be performed on different materials such as cultured cells, tissue sections, tissue microarrays or TMA. The above Example 1 is optimized for high-throughput drug screening on a 384-well plate, but this method can be adapted to a 1536-well plate. With this example, the RIF-Seq method can easily be performed on a much smaller scale, involving a single or just a few wells or tissue sections. In this example, if a lower scale experiment is desired, the same RT reaction mixture is introduced into all wells. Since the wells receive the same reaction components, the RT primers do not contain well-specific barcodes, but separate PCR handles ( image 3 , Figure 4 B). RT reactions were performed with biotinylated dNTPs and mRNA was reverse transcribed (RT) in fixed cells in separate wells. rRNA naturally occurring in structured form or in ...
Embodiment 3
[0105] Components of embodiment 3-RT primers and the positioning of sequences in the sequencing library
[0106] As described above in Example 1, cDNA from individual wells can be barcoded by using unique RT primers. The assignment of primers can be done automatically, and then a common RT mix can be dispensed to all wells simultaneously. After ligation of common 3' adapters, the pooled cDNA can be further indexed with plate-specific sequences ( Figure 4 A). If the experiment was performed as described in Example 2, the samples were not pooled until the end of the procedure. Samples received the same RT primers and the same adapters. During the library extension step, where samples are indexed with the plate-specific sequences in Example 1, samples can be indexed by wells and plates according to the scale of the experiment and user choice. Well and plate indexes can be positioned on either side of the sequencing library ( Figure 4 B). At the level of the complete seq...
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