alias: GSM4873002
title: GSM4873002: mir139WT14d; Mus musculus; OTHER
library_construction_protocol: DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, 69506) according to manufacturer's protocol, with the addition of 400ug RNase A (Qiagen) per 5 million cells. To determine over or under representation of sgRNAs, the integrated lentiviral sgRNA constructs were amplified by PCR followed by a nested PCR as described with modifications described below (Sanjana et al., 2014). Briefly, the first PCR reaction was performed for each sample in 13 separate 100 µl reactions. Each reaction contained 10 µg genomic DNA to get an estimated 300 times complexity using High Fidelity Herculase II Fusion DNA Polymerase (600675, Agilent Technologies), dNTPs (Invitrogen) and 0.5 µM of primers sgRNAlibF1 and sgRNAlibR1 (Table XX). The sequences encoding the sgRNAs were amplified using the following PCR conditions: 2 min at 95°C, 20 cycles of (30 sec at 95°C, 30 sec at 55°C, 1 min at 72°C) and fragments were extended for 10 min at 72°C. All PCR reactions were pooled and mixed. The second one-sided nested PCR reaction was performed for each sample in 6 separate 100 µl reactions. Each reaction contained 5 µl of the crude product from the first PCR, Fusion polymerase, dNTPs and 0.5 µM of primers sgRNAlibF2 and sgRNAlibR2. The concentration and quality of the libraries were determined by Agilent 2100 Bioanalyzer using a DNA1000 assay. Cluster generation was performed according to the Illumina TruSeq SR Rapid Cluster kit v2 (cBot) Reagents Preparation Guide (Illumina). Briefly, pooled libraries (10nM) were denaturated with NaOH, diluted to 10 pM and hybridized onto the flowcell. The hybridized products were sequentially amplified, linearized and end-blocked according to the Illumina Single Read Multiplex Sequencing user guide (Illumina). After hybridization with a custom-made sequencing primer (CGTTGTTCTTGTGGAAAGGACGAAACACCG), sequencing-by-synthesis was performed using the HiSeq 2500 (Illumina) using a single read 50-cycle protocol followed by single index sequencing.
identifiers.namespace: GEO
identifiers.id: GSM4873002
attributes.tag: GEO Accession
attributes.value: GSM4873002