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  • ATAC-seq primers

    Hi guys,

    I'm a newbie about the ATAC-seq, maybe here there is someone more expert than me.

    I'm doing an ATAC-seq on my samples (I have 3 different samples form thymic epithelium), and I'm a bit stucked about how to amplify my tagmented DNA. I've seen the primers list from Greenleaf lab (Supplemental table from the article), but now I have no idea of how to use those primers. Do I have to use all of them? I need to mix them?

    Sorry if it's a stupid question. Science is infinite knowledge!

    thank you very much for your help.

  • #2
    you use a PCR Primer 1 (Ad1_noMX) and an indexed PCR primer 2 (Ad2.1_... or Ad2.2_...or any index you like from the list) for each of your sample.

    Use different index primers for different samples,so your samples can be sequenced together.

    Do not mix the primers.

    Comment


    • #3
      Hi guys,

      In the ATAC-seq protocol, they said you can do a qPCR with Nextra Custom Design primers 1 and 2, are these primers Ad1_noMX and indexed primer 2 (what we use initially to do library amplification?) Or are these primers part of the Nextra Kit (FC-121-1030). like generic primers?

      Thanks very much!!!

      Comment


      • #4
        Hi Hedgehog,

        I oredered primers from Sigma, and they worked fine!
        Ciao!

        Comment


        • #5
          Originally posted by Fasteno View Post
          Hi Hedgehog,

          I oredered primers from Sigma, and they worked fine!
          Ciao!
          Hello,

          Could you help me with the primers? I am trying to order it to Sigma, with the PCR Primer 1 (Ad1_noMX) I haven´t got problems because it is a normal primer but with the PCR primer 2 Ad2.1 that must be BARCORDE, could you help how I have to order the BARCODE of the primer?
          Thank you in advance. Your help would be very grateful

          =D

          Comment


          • #6
            Thank you so much,

            My email is [email protected]

            Comment


            • #7
              sigma has .2-1uM range for adapter sequences

              In atac-seq protocol they say 25uM of primer but sigma has .2-1um range. Can you tell how you ordered.Thanks,

              Comment


              • #8
                How to order ATAC-seq primers

                I am about to order the Nextera DNA prep kit (24 samples) from illumina.

                I also need to order the primers (the ones in Supplementary Table 1 in the Nat Protocols paper) but I am new at this and want to make sure i do it correctly... do these primers need to be specially ordered in any way (ie. HPLC purified, etc.) and what concentration should i request?

                Comment


                • #9
                  HI,

                  I'm a little confused how to orders the primers too:
                  Just to be sure if I understand:

                  For each sample:

                  I will need:

                  Sample1
                  Ad1_noMX:
                  Ad2.1_index

                  Sample2
                  Ad1_noMX:
                  Ad2.2_index:

                  etc
                  In terms to order the primers if I follow the list from (Jason D Buenrostro paper):

                  I should be order:

                  Ad1_noMX: AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG


                  Basically the second primer I have to put the index sequence to order:
                  index+sequence?
                  Ad2.1_TAAGGCGA: TAAGGCGA-CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT


                  Thanks

                  Comment


                  • #10
                    ATAC-seq primers

                    That is correct

                    Here, order these:

                    Purification - NGS-grade (although DESALT worked for me as well)

                    AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG - Ad1_i5_noMX
                    CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT - Ad2.1_TAAGGCGA

                    CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGT - Ad2.2_CGTACTAG

                    CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGT - Ad2.3_AGGCAGAA

                    CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT - Ad2.4_TCCTGAGC

                    CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGT - Ad2.5_GGACTCCT

                    CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGT - Ad2.6_TAGGCATG

                    Comment


                    • #11
                      Originally posted by kropak1807 View Post
                      That is correct

                      Here, order these:

                      Purification - NGS-grade (although DESALT worked for me as well)

                      AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG - Ad1_i5_noMX
                      CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT - Ad2.1_TAAGGCGA

                      CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGT - Ad2.2_CGTACTAG

                      CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGT - Ad2.3_AGGCAGAA

                      CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT - Ad2.4_TCCTGAGC

                      CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGT - Ad2.5_GGACTCCT

                      CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGT - Ad2.6_TAGGCATG
                      Just a quick question the final primer that you order is:
                      For example:

                      For:
                      Ad2.1_TAAGGCGA
                      - CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT

                      Did you add the index:
                      Basically, the primer sequence I have to order is:
                      This one for ad2.1:
                      TAAGGCGACAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT

                      or this one:

                      CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT


                      Thanks

                      Comment


                      • #12
                        ATAC-seq primers

                        No, you do not add the sequence at the beginning of the primer.

                        Look closely,

                        CAAGCAGAAGACGGCATACGAGAT[8mer_index]GTCTCGTGGGCTCGGAGATGT-3’

                        The 8mer_index is the Index sequence complement.

                        So, for the sequences I posted, they already have the 8mer index (complement) in them.

                        Order the primers exactly as I posted them. Use the Index ID in the primer name to demultiplex your reads.

                        Comment


                        • #13
                          Originally posted by kropak1807 View Post
                          No, you do not add the sequence at the beginning of the primer.

                          Look closely,

                          CAAGCAGAAGACGGCATACGAGAT[8mer_index]GTCTCGTGGGCTCGGAGATGT-3’

                          The 8mer_index is the Index sequence complement.

                          So, for the sequences I posted, they already have the 8mer index (complement) in them.

                          Order the primers exactly as I posted them. Use the Index ID in the primer name to demultiplex your reads.
                          Thanks a lot.

                          Comment


                          • #14
                            Originally posted by newmoon View Post
                            you use a PCR Primer 1 (Ad1_noMX) and an indexed PCR primer 2 (Ad2.1_... or Ad2.2_...or any index you like from the list) for each of your sample.

                            Use different index primers for different samples,so your samples can be sequenced together.

                            Do not mix the primers.


                            Hi guys,

                            I am new to the ATACseq, and I am confused with the primers too. Why the index primers have to be different for different samples? You said that for the samples to be sequenced together, but I don't understand that... So you amplify the different samples and then send to sequence as 1 sample because they have different indexes?

                            Thanks a lot

                            Comment


                            • #15
                              Hi guys,

                              I am new to ATAC-seq, I plan to try this method with Arabidopsis. I am going to order those primers from Supplementary Table 1 (Buenrostro et al., Nat Methods, 2013). For Ad1_noMX and bar-coded primers Ad2.1/2/3..., just order them as what they are? is there any modification for these bar-coded primers? I plan to order them with DESALT from Eurofins, could anyone give me some suggestions?

                              Thank you very much,

                              Jie

                              Comment

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