Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: For ChIP-seq, Briefly Drosophila S2 cells were crosslinked with 1% formaldehyde for 20 min at room temperature, after which crosslinking was quenched in 0.125 M glycine. Isolated nuclei were sonicated using Misonix 3000 Sonicator, and the supernatant used for immunoprecipitation with the indicated antibody. ChIP-seq libraries were then sequenced on the Illumina HiSeq 2500 platform. For RNA-seq, total RNA was extracted using the QIAGEN RNeasy mini kit. Libraries were then prepared using the Illumina TruSeq Standard mRNA LT sample prep kit, and sequenced on the Illumina HiSeq 2500 platform. For PRO-seq, nuclei was first isolated from S2 Cells, followed by run on and biotin enrichment. biotinylated transcripts were then cloned using 5' and 3' RNA adaptors. Cloned transcripts were then reverse transcribed, PCR amplified, and sequenced on the Illumina Nextseq 500 platform. For ChIP nexus, briefly, Cells were cross-linked in 1.0% formaldehyde, lysed and sonicated using the Bioruptor Pico. Sheared chromatin was used in immunoprecipitation with the indicated antibodies. While the DNA is still bound to the antibodies, the DNA ends were repaired, dA-tailed, and ligated to adapters with single 3'-T overhangs and 5' overhangs. Cloned fragments were blunted by Klenow 3'-5' exo-polymerase followed by end-trimming using T4 DNA polymerase. The blunt end DNA was digested with λ-exonuclease followed by RecJf. Single stranded DNA products were then eluted, reverse cross-linked and purified. Purified DNA was then circularized using Circligase, and linearized using BamH1 after addition of an oligonucleotide complementary to the BamHI restriction site. Linearized single stranded DNA was then PCR-amplified using adaptor sequences. Libraries were then gel purified and sequenced on the Illumina NextSeq 500 platform. ChIP-seq libararies were prepared using the KAPA HTP Library Prep Kit for Illumina and Bioo Scientific NEXTflex DNA barcodes. Libraries were purified using the Agencourt AMPure XP system (Beckman Coulter) then quantified using a Bioanalyzer (Agilent Technologies), a Caliper LabChip GX (Perkin Elmer) and a Qubit fluorometer (Life Technologies). Post amplification size selection was performed on all libraries using the SPRIselect system (Beckman Coulter). Libraries were re-quantified, normalized, pooled and sequenced on an Illumina HiSeq 2500 instrument as 50-bp single reads using HiSeq Control Software 2.2.58. RNA-seq libraries were generated from 1ug of high quality total RNA, as assessed using the Bioanalyzer (Agilent). For samples in the RNAseq_1 series, libraries were made according to the manufacturer's directions using the TruSeq Stranded mRNA LT Sample Prep Kit – set A (Illumina, Cat. No. RS-122-2101) kit. For samples in the RNAseq_2 series, libraries were made according to the manufacturer's directions using the TruSeq Stranded mRNA LT Sample Prep Kit - set A and B (Illumina, Cat. No. RS-122-2101 and RS-122-2102) kit. The resulting short fragment libraries were checked for quality and quantity using the Bioanalyzer (Agilent) and Qubit Fluorometer (Life Technologies). Libraries were pooled, re-quantified and sequenced as 50 bp single reads on the Illumina HiSeq 2500 instrument. PRO-seq libraries were generated essentially as described in Mahat et al (2016), Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc. 11, 1455-76. doi: 10.1038/nprot.2016.086. Primers RPI4, RPI5, RPI6 and RPI7 were used to barcode cDNAs from eGFPdsRNA_rep1 (Non-targeting control, replicate 1), eGFPdsRNA_rep2(Non-targeting control, replicate 2), SSRP1kda1 (SSRP1 dsRNA mediated knockdown, replicate 1), and SSRP1kda2 (SSRP1 dsRNA mediated knockdown, replicate 2) respectively. ChIP-nexus libraries: DNA was end repaired using NEBNext End repair module (#E6050L). A single 3'-A overhang was added to the blunted ends using NEBNext dA-Tailing module (#E6053L). Adapters with single 3'-T overhangs and 5' overhangs (barcode composed of 5 random nucleotides) were ligated to the adenylated fragments. Ligated fragments were blunted again by Klenow 3'-5' exo- polymerase to fill in the 5' overhang first and then by T4 DNA polymerase to trim any possible 3' overhang. Blunted DNA was subsequently digested by lambda exonuclease first and RecJf afterwards. Digested single strand DNA was then eluted, reverse cross-linked and purified prior to self-circularization by Circligase. An oligonucleotide was mixed with circularized single DNA for subsequent BamHI digestion to linearize the single DNA again. Linearized single strand DNA was then PCR-amplified using adaptor sequences and library was purified on 2% agarose gel to remove adaptor-adaptor ligation products.