title: GSM8122748: overhang_screen_6mer_rep1; synthetic construct; OTHER
library_construction_protocol: No extraction protocol, starting DNA oligonucleotides were synthesized with desalting purification and used as received Eight oligonucleotides (BL358 to BL365) were synthesized with normal desalting purification. The oligos were annealed to produce two double-stranded DNA molecules (A and B) containing a constant region corresponding to the truseq P5 and P7 sequences plus a multiplexing sequencing indes, and a random 6-mer or 7-mer sequence protruding at the 5' end. Oligos were annealed at 30 uM concentration in 2X SSC by running the following program in a thermocycler: 1) 95C for 5 minutes, 2) ramp down to 65 at 0.1C/second, 3) hold at 65C for 5 minutes, 4) ramp down to 12C at 0.1C/second, 5) hold at 12C. The A and B dsDNA molecules for each overhang length (6-mer or 7-mer) were diluted to 3.334 uM in 2X SSX and used to assemble a ligation reaction as follows: 2 uL 10x T4 ligase buffer, 1 uL of each annealed dsDNA (final 166.7 nM), 1 uL T4 DNA ligase (NEB M0202M), 15 uL nuclease-free water. Ligation was performed for 3 minutes at 22C, following by heat-inactivation at 65C for 10 minutes. The ligated molecules were purified by running the whole reaction volume on a 2% TBE agarose gel. The gel was stained in 1:10000 SYBR-gold (Thermo Scientific) and the bands corresponding to the ligation products were purified using the Qiagen Qiaquick gel extraction kit using the supplier's standard protocol. Library concentration was estimated by tapestation (DNA5000 screentape) and qPCR (KAPA Illumina library quantification kit)