alias: GSM3375591
title: GSM3375591: DT40_SNS-seq; Gallus gallus; OTHER
library_construction_protocol: SNS were double-stranded by random priming with the Klenow exo-polymerase (# EP0421, Thermo Fisher Scientific) and random primers (#48190011, ThermoFisher Scientific). Adjacent strands were then ligated with Taq DNA Ligase (M0208L, Biolabs). RNA primers were hydrolysed for 30 min at 37oC with 2.5 M NaOH. The reaction was then neutralized with 2.5 M acetic acid, and SNS were purified by phenol chloroform extraction and recovered after ethanol precipitation and resuspension in TE buffer. SNS molecules were quantified with the QBit dsDNA HS assay kit (Q32854, Thermo Fisher Scientific) and 225 ng of double-stranded material was used for fragmentation using a Covaris apparatus with a 50-μl microTUBE-50 AFA Fiber screw-cap tune (PN 520166, Covaris) and a M220 Holder XTU (PN500488, Covaris). We used a soni- cation programme with 75 W peak incident power, 10% duty factor and 200 cycles per burst running at 4o C for 195 s to obtain molecules with a size of approximately 180 bp. The library was constructed with the NEBNext©UltraTM II DNA Library Prep Kit for Illumina©(NEB#E7645S) following the manufacturer's instructions with some minor modifications. For the adaptor ligation, undiluted adaptor and no size selection were used. The library amplification The library amplification was performed using NEBNext ©Multiplex Oligos for Illumina©(NEB #E7335S) with NEBNext index 4 primer (#E7314A) and the NEBNext Universal PCR Primer (#E6861A) with three PCR cycles. Library purification was performed with the SPRIselect Reagent kit (Beckman coulter #B23317), and the final elution step was reduced to 20 μl of 0.1X TE. The mean size of the library molecules determined on an Agilent Bioanalyser High Sensitivity DNA chip (5067- 4626, Agilent technologies) was 550 bp. Sequencing was performed on a NextSeq 500 Illumina sequencer with a High Output 150 cycles flow cell (paired-end reads of 75 bp) according to standard procedures. A total of 246,14 M clusters were generated and provided 492,28 M of reads. All Illumina sequencing runs were performed at the GENOM'IC facility of the Cochin Institute.
identifiers.namespace: GEO
identifiers.id: GSM3375591