title: GSM8903354: LCR HS combination library, 0,3,10,20,50,100kb, HBG promoter rep1, bin3; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was extracted from each sorted population using Qiagen DNA mini kit (Qiagen, #51304). For each of the genomic DNA sample, we performed 8 separate 50ul reactions with lr_P3:  GCATGCGAATGGCCTCGTAG and lr_P5: CACAGCCAGACCAACATTGCC using the following condition: 98 °C for 30 s; 20 cycles of 98 °C for 15 s, 69 °C for 15 s and 72 °C for 45 s. After PCR, we pooled eight PCRs for each sample, cleaned up DNA using 1.2x SPRIselect beads(Beckman Coulter, B23317) and quantified DNA concentration using Qubit (Invitrogen, Q33231). We added sequencing adapters and indices with two additional rounds of PCRs: for PCR2, we took 4ng PCR1 product as template and performed 8 cycles of PCR with primers lr_P4: CTTTCCCTACACGACGCTCTTCCGATCTGCATGCGAATGGCCTCGTAG and lr_P6: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCACAGCCAGACCAACATTGCC. For PCR3, we took 2ul PCR2 product as template and amplified another 8 cycles using primers IDT_10_i5: AATGATACGGCGACCACCGAGATCTACAC(10bp index)ACACTCTTTCCCTACACGACGCTC and IDT_10_i7: CAAGCAGAAGACGGCATACGAGAT(10bp index)GTGACTGGAGTTCAGACGTG. After PCR3, we cleaned up DNA using 1.2x SPRIselect beads and quantified DNA concentration using Qubit. (Invitrogen, Q33231).
identifiers.namespace: GEO
identifiers.id: GSM8903354_r1