P-GSE41187-3 - P-GSE41187-3
nucleic acid library construction protocol
Antibodies for beta of RNAP - NeoClone, Cat Number: W0002, Lot Number: 2008L10-001 Antibodies for sigma70 of RNAP - NeoClone, Cat Number: W0004, Lot Number: 2008K12-001 All other antibodies (FNR, H-NS, IHF) were produced in this study and are not commercially available. 10 ng of DNA were submitted to the University of Wisconsin-Madison DNA Sequencing Facility for ChIP-seq library preparation. Samples were sheared to 200-500 nt during the IP process to facilitate library preparation. All libraries were generated using reagents from the Illumina Paired End Sample Preparation Kit (Illumina) and the Illumina protocol “Preparing Samples for ChIP Sequencing of DNA” (Illumina part # 11257047 RevA) as per the manufacturer’s instructions, except products of the ligation reaction were purified by gel electrophoresis using 2% SizeSelect agarose gels (Invitrogen) targeting either 275 bp fragments (s70 libraries) or 400 bp fragments (FNR libraries). After library construction and amplification, quality and quantity were assessed using an Agilent DNA 1000 series chip assay (Agilent) and QuantIT PicoGreen dsDNA Kit (Invitrogen), respectively, and libraries were standardized to 10μM. Cluster generation was performed using a cBot Single Read Cluster Generation Kit (v4) and placed on the Illumina cBot. A single read, 36 bp run was performed, using standard SBS kits (v4) and SCS 2.6 on an Illumina Genome Analyzer IIx. A paired read, 100 bp runs were used for one replicate of each s70 aerobic and anaerobic growth conditions, using standard SBS kits (v4) and SCS 2.6 on an Illumina HiSeq. Basecalling was performed using the standard Illumina Pipeline version 1.6.