early batch 1 R2
Homo sapiens
Protocols: Two pools of human iPSC WTC11 transfected with a pool of iPS2-seq shRNA library were cultured for different passages, and the cell pellets were frozen. Collections were performed at early passage (P3 early), then after five passages (P3+5 mid), and another five passages (P3+10 late). Cells were cultured on Matrigel and mTseR plus. Genomic DNA was extracted from human cell lines at various passages and timepoints using the Monarch® Spin gDNA Extraction Kit (NEB #T3010) and following the user guide SD2163380 and section \"Genomic DNA Extraction 1 protocol for PCR applications\". Samples samples eluted in 70 μL elution buffer. DNA purity was qunatified by nanodrop and assessed by A260/A280 and A260/A230 ratios, ensuring quality suitable for downstream applications. Each extracted sample was quantified by Qubit fluorometry and TapeStation (D1000), and normalized to approximately 500 ng total input per PCR reaction, with adjustments made where DNA concentration was limited. PCR reactions were prepared using the Q5 High-Fidelity DNA Polymerase system, with a master mix containing 5X Q5 Reaction Buffer (NEB), dNTPs (NEB), and custom primers. Each reaction included 27 μL master mix and appropriate volumes of template DNA and nuclease-free water to achieve a final volume of 50 μL. PCR cycling conditions were: initial denaturation at 98°C for 30 seconds; 26 cycles of 98°C for 10 seconds, 64°C for 30 seconds, and 72°C for 20 seconds; and a final extension at 72°C for 2 minutes. Amplified products (~144 bp) were purified using SPRIselect beads (beckman) at 0.6X and 1.5X bead ratios to remove primer dimers and small fragments. Purified PCR products were diluted to 0.1 ng/μL in EB buffer prior to indexing PCR. Indexing was performed using the NEBNext High-Fidelity 2X PCR Master Mix with dual-index primers (10X plate TT set A) in 20 μL reactions (10 μL master mix, 4 μL dual-index primers, 1 μL PCR product, and 5 μL water). The PCR conditions included: initial denaturation at 98°C for 30 seconds; 10 cycles of 98°C for 10 seconds, 58°C for 20 seconds, and 72°C for 20 seconds; followed by a final extension at 72°C for 30 seconds. Indexed libraries (~228 bp) were purified using a double-sided SPRIselect bead cleanup (0.65X and 1.5X ratios). Final libraries were quantified by TapeStation D1000 and Qubit dsDNA HS assays. Indexed libraries were then pooled with other libraries to allow higher complexity and prepared for sequencing.
ENA first public
2025-12-29
INSDC center name
UniTo
INSDC status
public
Submitter Id
E-MTAB-15303:early batch 1 R2
broker name
ArrayExpress
cell line
WTC11
cell type
iPSC
collection date
not collected
common name
human
developmental stage
early pasage iPSC
disease
normal
genotype
iPS2seq transfection pool 1
geographic location (country and/or sea)
not collected
growth condition
p1
isolate
not applicable
sample name
E-MTAB-15303:early batch 1 R2
scientific_name
Homo sapiens