1. An RNA-seq profiling experiment is a different approach to getting the inform
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1. An RNA-seq profiling experiment is a different approach to getting the information from a footprint. In this experiment, an RNA is bound by a strongly binding complex such a ribosome or an snRNP. Then an RNase is used to digest away all of the unprotected RNA. The undigested RNAs get linkers added to both ends and are then converted into cDNAs. The cDNAs are sequenced by Illumina sequencing. Draw out all the steps to show how this method would be used to determine the RNA that is bound by the U2-U2AF65/35 complex. Indicate what linker sequences are used for. Specify primers, and describe any additional processing needed for the Illumina sequencing.Explanation / Answer
start with 0.5 ?g of DNAse-treated human total RNA in the first step of the TruSeq RNA Sample Preparation v2 protocol (Illumina, Part# 15026495 Rev. A). But any amount of total RNA between 0.1 and 4 ?g of total RNA, as recommended in the Illumina protocol can be used. Following reagents are required:
•TruSeq RNA sample preparation Kit v2, Set A (Illumina, #RS-122-2001).
•illustraMicroSpin G-50 Columns (GE Healthcare; #27-5330-02).
•1 mM Tris pH 8.0 (dilution from 1 M Solution, Ambion, #AM9855G).
•Elution Buffer (Qiagen, #19086).
•10× Reverse Transcription Buffer (Invitrogen, #53032).
•5× Second Strand Synthesis Buffer (Invitrogen, #11917-010).
•100 mM DTT (Invitrogen, #11917-010).
•E.Coli DNA ligase (10 U/?l) (NEB, #M0205L).
•DNA Polymerase I (10 U/?l) (NEB, #M0209L).
•RNase H (2 U/?l) (Invitrogen, #100004927).
•100 mM MgCl2 (Dilution from 1 M solution, Ambion, # AM9530G).
•dUNTP Mix (10 mM each dATP, dCTP, dGTP, dUTP) (Fermentas; #R0146, #R0156,#R0166, #R0133).
•UDGase (1 U/?l) (NEB, #M0280S).
•10× UDG Buffer (NEB, #B0280S).
•RNase free water
The first steps (1) Purify and Fragment mRNA and (2) First Strand Synthesis were performed as described in the Illumina kit. Step (3) Second Strand cDNA Synthesis was modified as follows:
1.Spin illustra MicroSpin G-50 Columns at 700×g for 1 min.
2.Wash Columns three times with 1 mM Tris–HCl pH 8.0:
i.Add 500 ?l Tris–HCl to the column and resuspend the resin by gentle mixing.
ii.Centrifuge column at 700×g for 1 min.
3.Bring the column into a 1.5 ml Low Binding tube.
4.Add 5 ?l Elution Buffer to the sample.
5.Add the sample (30 ?l) to the G-50 Column and spin the column at 700×g for 2 min.
6.Measure the volume of the eluate (should be between 30 and 50 ?l).
7.Add RNAse free water to the sample up to a total volume of 52.5 ?l.
8.Add Second Strand Mix (22.5 ?l) to the sample:
i.Second Strand Mix: 1 ?l of 10× Reverse Transcription Buffer, 15 ?l of 5× Second Strand Syntheses Buffer, 0.5 ?l of 100 mMMgCl, 1 ?l of 100 mM DTT, 2 ?l of dUNTP Mix, 0.5 ?l of E. Coli DNA ligase, 2 ?l of DNA Polymerase, and 0.5 ?l of RNase H.
9.Incubate Mix at 16 °C for 2 h.
10.After incubation add 135 ?l Ampure XP Beads to the Mix (instead of the 90 ?l described in the TruSeq Protocol) and vortex the tube.
11.Continue with the TruSeq Protocol at the incubation step (15 min at RT; point 3 of page 87 of the Illumina protocol) of “Clean Up CDP” of the Second Strand Synthesis.
12.Perform End Repair, Adenylate 3? Ends and Adapter Ligation steps as described in the TruSeq Protocol.
13.After “Adapter Ligation” and before starting “Enrich DNA Fragment step, perform the the UDGase Treatment by adding 2.3 ?l of 10× UDG Buffer and 1 ?l of UDGase (1 U/?l) to the sample.
14.Incubate the sample for 30 min at 37 °C.
15.Continue with the “Enrich DNA Fragment” step and follow the TruSeq protocol until the end.
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