[No authors listed]
The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.
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AKT3, LOC100129973, PP14571, LOC100131510, SH3RF3-AS1, ZNF783, RBFADN, LOC100506472, LINC01007, LOC100506639, LOC100507424, ABCB6, LINC00563, ARPC1B, LRPPRC, PDIA6, OPTN, PLXNC1, WASF2, CASC20, RNF41, CDK4, STX6, ATP8A1, RAPGEF3, UBAC1, LYPLA1, OLFM1, CARM1, SEMA6C, DDX17, LOC105377443, AGPAT1, STAMBP, FAM189B, AHCYL1, SRSF10, ADCY2, MALT1, JTB, RUNDC3A, MORF4L1, EHD1, YWHAQ, FAM107A, DDX20, PACSIN2, ATXN2L, LARP4, EXOC3, RNF13, MGLL, OSBPL11, MARCH3, KCTD12, CLCN4, C14orf79, SPATA2L, ALDH16A1, MBOAT2, COX4I1, COX11, CPD, UBXN2B, RALYL, CRYAB, HECTD2, CSK, SLC38A6, CSNK1G2, BEAN1, TOM1L2, B3GNTL1, WIPF2, TICAM1, C19orf25, ZNF555, NBPF12, CNIH3, CTNND2, SPICE1, LOC155060, ANKRD46, LINC00599, CD55, DBP, PLPPR5, DFNA5, ABHD3, DHPS, DIAPH1, DIO2, DNM1, DOK1, RCAN1, TSC22D3, DTNB, EIF4A1, TXLNA, SPRED2, EML1, SAMD14, HACD2, PDE12, ENO1, TUBB, C9orf91, FAM219A, EPAS1, EPB41L2, STX2, EPS15, ERBB4, ETV1, EYA2, FAM13C, SDK1, RHOBTB3, VASH1, NLRP1, DIS3, SIRT2, SHANK2, EFR3B, WDFY3, KDM4B, ENDOD1, ALDOC, PEG10, NFASC, TBC1D9, PASK, RCOR1, FAM120A, ACSBG1, KIAA1024, KAZN, NCAPD3, KIAA0930, NEDD4L, UBR4, OBSL1, KIAA0368, DICER1, SF3B1, ANGPTL2, NPTXR, KCTD2, MAPK8IP2, FAM89B, STX12, PISD, FLRT1, FUT8, ASPHD1, LPCAT4, ZDHHC23, KANK3, C2orf72, SUN2, FAM19A5, NBPF14, DCAF12, PART1, GAPDH, TBC1D10B, LRP10, ZNF500, TRPC4AP, SERBP1, FBXL5, BLOC1S6, SEZ6L2, BSCL2, GFPT1, TRUB2, KCNV1, CNPPD1, NPTN, GHITM, B3GAT1, TRAPPC3, ARRDC2, ATP5S, SDCBP2, DKK3, GLS2, AHDC1, ERLEC1, MMADHC, CSDC2, PCDH11X, DESI1, EML4, BIN1, GNAI1, GNB1, GOT1, GPD2, CCDC57, LOC284244, ANK2, GRN, HIPK2, PHPT1, GRM5, SLC25A4, SLC25A6, GTF3C2, C2orf27A, SAC3D1, FAM216A, CCDC106, SCHIP1, SLC39A3, HELLS, TAX1BP3, ACAA1, HLA-B, HLF, HNRNPH3, APBA2, HPCAL1, APC, PRMT2, APEX1, HSPB1, HTR6, OXLD1, LINC00667, TUBB2B, IGFBP5, NIPAL4, IGHMBP2, IK, IL15RA, ILF3, ARAF, IVD, GALNT18, STUM, ARF1, KPNA1, KPNA4, CEP85L, C6orf120, FADS1, ARL3, LY9, MAP4, MCM7, MGAT5, MMP16, PRKCQ-AS1, CCDC88C, LOC441052, NARS, NF1, NMT1, ATP2B1, NTRK3, OPCML, P2RX5, CHST11, ITSN2, PAK3, PAM, WDPCP, ZNF691, FAM135B, TXNDC12, TXNDC11, ATL1, CALHM2, MRPL4, POLR1D, UTP18, ABHD5, NAGPA, MARCH2, BET1L, ZDHHC3, REEP2, ANAPC7, IP6K2, EVL, CXXC5, VTA1, RAB14, AZIN1, ASB1, SELT, TMEM8B, MYOZ2, PGAM1, PIK3C3, PKD2, PLTP, PTOV1, COPG2IT1, ACP2, POLE3, KCNK10, POLR2B, ATAD2B, MRPS21, CCSER2, MIEF1, FAM105A, TMCO1, DHX29, HMGCLL1, CCDC93, FAM35A, NECAB2, CROT, KIF1A, WBP1L, LRRC8D, RIF1, VPS13D, LGI2, PHF10, RFK, C5orf22, PI4K2A, MEG3, ZNF821, SMPD4, BTBD2, OSGEP, MFN1, MAP7D1, IPO9, DNAJC11, VPS35, PRKAR1A, EDEM2, NUP133, ZNF415, FOXJ2, WSB2, RNF114, AJAP1, MAPK10, PCDHB10, MTFR1L, HTRA1, B2M, KCMF1, MDM1, HMCES, SMYD2, C5orf15, C14orf132, BBX, CDC42SE2, PSMC5, PNPLA2, PLXDC1, TMEM159, SLC44A2, SMURF1, ZMIZ1, NIPAL3, RALGAPA2, SPRYD7, MRS2, SLC12A5, MARCH4, POGK, BRINP2, PTPN11, ZNF410, ARHGAP22, CACTIN, DLGAP3, RAB2A, RAB6A, RAB27B, KDM5A, RFNG, RGS3, AVPI1, TGIF2, ELOVL5, BCS1L, RXRA, SCO1, SEC13, SEC14L1, RRAGC, C17orf75, SETMAR, MMS19, SRSF5, MPP5, CDH22, MTMR14, MEAF6, C6orf106, ST6GAL1, AIDA, USP46, C12orf43, NT5DC2, NDRG4, ACD, SLC1A2, SLC1A3, MARCKSL1, SLC2A1, SLC3A2, NADK, PAX8-AS1, SLC16A2, MTMR9, SNX1, SORL1, SPP1, SRD5A1, SRP9, STAR, SUPT5H, SUPT6H, TAPBP, TBCD, TCEB3, PPP1R11, TIAM1, TIMP3, TJP1, TMOD1, TMPO, TP53, LOC728392, OIP5-AS1, RRN3P1, UBC, USF2, NELFA, YWHAH, ZIC1, CACNA1C, CSDE1, ERI3, FTO, TMEM109, WDR77, TMUB2, DERL1, ZXDC, CAAP1, LPCAT1, ADCK4, SPSB1, TMEM134, BRD3, GRPEL1, FLAD1, PDHX, MLF2, BHLHB9, GRIP2, ANP32A, FAM49A, C1orf21, NDEL1, CSRNP2, NIPA2, CDT1, UNC93B1, CAMK2B, ISCA1, ST6GALNAC5, LZTR1, CANX, USP9X, CAPNS1, COG3, TLN2, CALN1, SH3BGRL2, NCALD, FAM172A, SLC25A11, TAGLN2, STX7, GNPAT, CUL3, NTNG2, OGT, MFSD9, SPRYD3, RITA1, MIR22HG, ARHGAP19, PIK3R3, DIXDC1, CAV1, PLPP3, RUNX1T1, AOC3, CHRD, TNKS, VAMP4, HYAL2, ADAM23, NAPG, SUCLG2, EIF2B2, CACNA1H, HERC1, FAM127A, BAIAP3, TOP3B, C5orf30, FAM110B, IFT20, ADCK2, DHX57, KIFC2, CREB3L1, AIFM1, TMEM257, UBXN11, ATPAF2, ZNF804A, COG7, DCLK1, XPR1, CYTH1, TMEM183A, ITGB1BP1, SHKBP1, MED14, IMP4, LINC00950, TEX30, OLFM2, GTF3C5, MAPK1IP1L, ARHGAP12, MAPK8IP1, CHST10, PSMF1, SCAMP1, TP53I11, EI24, ABCG1, MICAL2, RAPGEF2, RIMS2, HERPUD1, RAB11FIP3, HDAC4, KIAA0513, TMEM94, ATG13, MATR3, CEP350, SNAP91, SV2B, G3BP2, IQSEC1, DMTF1, PPP4R1
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