Sequencing Methods VEB

CAGE vs mRNA RNA-SEQ

TruSeq Stranded mRNA RNA-SEQ The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM), followed by second strand cDNA synthesis using DNA Polymerase I and RNase H

TruSeq Small RNA-SEQ The RNA 3' adapter is specifically modified to target microRNAs and other small RNAs that have a 3' hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes The adapters are ligated to each end of the RNA molecule and an RT reaction is used to create single stranded cDNA. The cDNA is then PCR amplified using a common primer and a primer containing one of 48 index sequences.

TruSeq Stranded Total RNA RNA-SEQ The first step involves the removal of ribosomal RNA (rRNA) using biotinylated, target-specific oligos combined with magnetic beads ( ..depletes samples of both cytoplasmic and mitochondrial rRNA …depletes globin-encoding mRNA ). Following purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H.

CHROMATIN ISOLATION BY RNA PURIFICATION (CHIRP-SEQ) Samples are first crosslinked and sonicated. Biotinylated tiling oligos are hybridized to the RNAs of interest. The complexes are captured with streptavidin magnetic beads. After treatment with RNase H the DNA is extracted and sequenced.

CHROMATIN ISOLATION BY RNA PURIFICATION (CHIRP-SEQ)

GLOBAL RUN-ON SEQUENCING (GRO-SEQ) Active RNA Polymerase is allowed to run on in the presence of Br-UTP.. RNAs are hydrolyzed and purified using beads coated with Brd-UTP antibody. The eluted RNA undergoes cap removal and end repair prior to reverse transcription to cDNA. cDNA is sequenced. cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

GLOBAL RUN-ON SEQUENCING (GRO-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

RNA IMMUNOPRECIPITATION SEQUENCING (RIP-SEQ)/CROSSLINKING AND IMMUNOPRECIPITATION SEQUENCING (CLIP-SEQ) RNA-protein complexes are immunoprecipitated with antibodies targeted to the protein of interest. RNase digestion and RNA covered by protein is extracted. The eluted RNA undergoes end repair prior to reverse transcription to cDNA. cDNA is sequenced. RNA-protein complexes is stabilized by Crosslinking. RNA-protein complexes are immunoprecipitated with antibodies targeted to the protein of interest. The protein-RNA complexes are treated with RNase followed by de-crosslinking and Proteinase K. The eluted RNA undergoes end repair prior to reverse transcription to cDNA and sequencing.

RIP-SEQ CLIP-SEQ cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

BISULFITE Genotyping cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

BISULFITE SEQUENCING (BS-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

BISULFITE SEQUENCING (BS-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING (MEDIP-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING (MEDIP-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

REDUCED-REPRESENTATION BISULFITE SEQUENCING (RRBS-SEQ) Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.Boyle P, Clement K, Gu H, Smith ZD, Ziller M, Fostel JL, Holmes L, Meldrim J, Kelley F, Gnirke A, Meissner A. Genome Biol. 2012 Oct 3;13(10):R92. cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

REDUCED-REPRESENTATION BISULFITE SEQUENCING (RRBS-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

CIRCULAR CHROMATIN CONFORMATION CAPTURE (4-C OR 4C-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

Overview of 3C-derived methods. Overview of 3C-derived methods. An overview of the 3C-derived methods that are discussed is given. The horizontal panel shows the cross-linking, digestion, and ligation steps common to all of the “C” methods. The vertical panels indicate the steps that are specific to separate methods. de Wit E , and de Laat W Genes Dev. 2012;26:11-24

CIRCULAR CHROMATIN CONFORMATION CAPTURE (4-C OR 4C-SEQ) cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.