title: GSM1255520: pooled HD3 rep 2 [4C-Seq]; Gallus gallus; OTHER
library_construction_protocol: PCR reactions were performed using the Expand Long Template PCR system (Roche). The 50 µl PCR reaction contained 120 ng of the DNA template, 1× PCR buffer 1, 0.3 µM of each of the primers, 0.35 mM of each of dNTPs, and 3.75 units of Hot start Taq DNA polymerase (SibEnzyme)Expand Long enzyme mix (Roche). The sequences of HindIII / DpnII 4C primers are as follows (5’-3’): TSR3 bait CACTCATCTCCCCGTACTTTG / AAGTTTCTTTTAATTTGGAGACTTTC; GENE-Des bait AATTTGTGAAGCAGTTGTATGTAGTC / TCTTCTCCACATAATCCCACACT; NPRL3 bait GCCAGGATATAGATTCTGCTTT / CCTCTGACATAATTGCCGATAG; TRAP1 bait CCAGAGATTCTCAAATCACAGCA / CTATGGGGACAAGTGAGGAACAG; PPL bait AAAGCATCTCCTCTCCCTGAAG / GTCTCCCACAGTCACTCCTCCT. PCR amplification was performed in the linear range as follows: initial denaturation for 2 min at 94°C; 30 cycles of 15 s at 94°C, 1 min at 57°C, and 3 min at 68°C; final elongation for 4 min at 68°C. PCR products were purified and concentrated using QIAquick PCR purification kit (Qiagen). After separation according to sizes in 1.5% agarose gel, two zones - 70-400 bp (S fraction) and 400-1500bp (L fraction) - were cut. After extraction with QIAquick gel extraction kit (Qiagen), the fragments of L-fraction were sonicated to reduce the fragment size to 100-500 nucleotides using Covaris S220 with following parameters: time 300 seconds, duty cycle 10%, peak power 23W. Then S and L fraction were processed using TruSeq DNA sample prep kit v.2 (Illumina) with post-PCR size selection on agarose gel (fragments with length 200-600 nucleotides were selected). After purification library concentrations were measured using Qubit fluorimeter (Invitrogen) and real-time PCR with primers complementary to distal regions of Illumina adapters (I-qPCR-1.1: AATGATACGGCGACCACCGAGAT and I-qPCR-2.1: CAAGCAGAAGACGGCATACGA). Then libraries from S and L fractions were combined in equal amounts, diluted to 2nM, denatured using 0.1M NaOH and diluted to final concentration 10 pM using HT1 buffer (Illumina). Cluster generation was performed using cBot instrument and TruSeq PE Cluster Kit v3 reagents and sequencing - using HiSeq2000 instrument and TruSeq SBS Kit v3 reagents (Illumina) with read length 101 nucleotides from each end.
attributes.tag: GEO Accession
attributes.value: GSM1255520