alias: GSM3529670
title: GSM3529670: Mix 367 sgRNA pool: 293T CATE-seq; Homo sapiens; OTHER
library_construction_protocol: Cells were inoculated into a 25 cm2 culture flask and cultured in complete medium for 24 h (degree of fusion greater than 80%). Cells were then washed twice with pre-chilled PBS and collected by trypsinization. The genomic DNA (gDNA) was extracted from cells by using the TIANamp Genomic DNA Kit (Tiangen). The extracted gDNAs of 293T, HepG2, HL7702, C-33a, SiHa, and HeLa cells were tagmented by the Tn5 transposomes T-B-1 to T-B-6, respectively. Additionally, the 293T gDNA was also tagmented by the Tn5 transposome T-B-7, which was called as 293Tm. The tagmentation reaction (30 μL) contained 200 ng gDNA, 5 μL transposome, 6 μL 5× LM buffer, and certain volume of ddH2O that supplemented the total reaction to 30 μL. The reaction was incubated at 55°C for 15 min. The reaction contained no transposome was used as control. The tagmentation products were run with agarose gel and the fragments of 200–1000 bp were recovered, which were used as the input DNA for enriching target sequences.The dCas9 protein was diluted to 0.3 μM with stock solution and stored at -20° C. The csgRNAs were mixed into four groups according to their numbering. In each mixing, about 7.5 ng of each csgRNA was used. Five μL of magnetic beads with streptavidin was added to 50 μL of PBS containing 0.5% BSA and placed on a magnetic stand for 15 s. The supernatant was aspirated. After washing three times with 50 μL of PBS-BSA solution, the beads were added with 50 μL of 1×dCas9 buffer and 2 μL of capture oligonucleotide (10 μM). The sequence of capture oligonucleotide was 5′-Biotin-TTTTTTTGCA TCTGGTATTC GTAAGGTTCC G-3′. The beads were mixing by rotation for 1 hour. The beads were washed three times with 50 μL of 1×dCas9 buffer.
identifiers.namespace: GEO
identifiers.id: GSM3529670