Primary naïve B cells isolated from Smc3wt/wt (n=3) or Smc3wt/– were cultured ex vivo to produce iGCs as explained (13 (link)). Genomic DNA was used to produce whole genome sequencing libraries using the KAPA LTP Library Preparation kit following manufacturer’s directions. Sequencing was done in NextSeq500 instrument using a 75 bp single-read sequencing cell. We used TIGER (20 (link)) to infer DNA copy number values at 1Kb windows in mm10 coordinates. TIGER separates continuous and low-amplitude signals of DNA replication timing from the larger and sharper changes caused by copy number alterations. For genome-wide visualization of raw DNA copy number values, every 40 consecutive windows were merged. For DNA replication timing, outlier segments representing putative copy number alterations were filtered out by TIGER, and the remaining data was smoothed, normalized to units of standard deviation, and plotted.
Ltp library preparation kit
Manufactured by Roche
The LTP Library Preparation kit is a lab equipment product designed for nucleic acid library preparation. It facilitates the construction of sequencing-ready libraries from various input samples. The kit provides the necessary reagents and protocols to prepare libraries for high-throughput sequencing applications.
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Lab products found in correlation
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Miseq platform, by Illumina (1 mentions)
Kapa library quantification kit, by Thermo Fisher Scientific (1 mentions)
M220 ultrasonicator, by Covaris (1 mentions)
Kapa pure beads, by Roche (1 mentions)
Kapa s hifi enzyme, by Roche (1 mentions)
Tapestation, by Roche (1 mentions)
Hc 20, by Santa Cruz Biotechnology (1 mentions)
3 protocols using ltp library preparation kit
1
Genome-wide DNA Copy Number and Replication Timing Analysis of Smc3-Deficient iGCs
2
Illumina DNA Library Prep for Sequencing
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Illumina compatible Genomic DNA libraries were generated on the Apollo 384 liquid handler using KAPA Biosystem’s LTP library preparation kit (KK8232). DNA was sheared to approximately 600 bp fragments using a Covaris M220 ultrasonicator, end repaired, and A-tailed as described in the Kapa protocol. Illumina-compatible adapters with unique indexes (IDT #00989130v2) were ligated on each sample individually. The adapter ligated molecules were cleaned using Kapa pure beads (Kapa Biosciences, KK8002) and amplified with Kapa’s HIFI enzyme (KK2502). Each library was then analyzed for fragment size on an Agilent’s Tapestation and quantified by qPCR (KAPA Library Quantification Kit, KK4835) on Thermo Fisher Scientific’s Quantstudio 5. Libraries were then multiplexed and sequenced on 2 × 250 flow cell on the MiSeq platform (Illumina) at the ASU’s Genomics Core facility.
3
Deciphering Transcription Factor Interactions
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T47D cells were treated for 45 min with ethanol or E2 plus R5020. ChIP was then performed using anti-ER (Santa Cruz Biotechnology, HC-20) as described above; however, chromatin was eluted in reChIP elution buffer (1× TE, 2% SDS, and 15 mM dithiothreitol supplemented with protease inhibitors). The eluted anti-ER ChIP sample was diluted 1:20 in ChIP dilution buffer supplemented with 50 μg of bovine serum albumin and protease inhibitors. The secondary ChIP (reChIP) was performed using anti-PR (KD68) or anti-IgG. Primer sequences used for reChIP–qRT-PCR (quantitative RT-PCR) are provided in table S2. For reChIP-seq library preparations, the final reChIPed DNA fragments were reconstituted in 30 μl of nuclease-free water. DNA (15 μl) was used to prepare reChIP-seq libraries using Kapa Biosystems LTP Library Preparation Kit (no. KK8232) according to the manufacturer’s protocol. For the PCR enrichment step of the library preparation protocol, 12 PCR cycles were performed.
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