to design shrnas against cd antigens , we used the shrna prediction algorithm freely available through the hannon lab website : http://katahdin.cshl.org:9331/homepage/portal/scripts/main2.pl15 .
the output shrna sequences were then modified to have a 9-bp loop ( ttcaagaga ) and common primer binding sites compatible with cloning into the lentiviral vector psicor - mcherry which drives stable expression of the hairpin via a mouse u6 promoter16 : 5 ' : cgcctgcgagtctggtatg , 3 : ggaattcgccagctcgag ( reverse complement ) .
a 22,000-element , 96-bp oligonucleotide pilot library was designed using these sequences , and was provided by agilent technologies as a pool in a single tube ( 4 pmol ) and dissolved in 200 l water .
improvements to fidelity of long oligonucleotides were made by minimizing the synthetic cycle loss during coupling and deblock , and by minimizing the depurination side reaction .
we identified optimal conditions for amplification of full - length product ( 96 bp oligos ) by using phusion dna polymerase ( finnzymes ) , varying the amount of dmso , annealing temperature , and extension times for the pcr on a bio - rad icycler .
final conditions were for 50 l reaction : 30 l water , 10 l 5x phusion gc buffer , 1 l 10 mm dntps , 5 l 5 m primer mix , 0.5 l template , 1 l dmso , 0.5 l hot start phusion polymerase ( finnzymes ) .
cycling parameters were 98 c for 30 s ; 15 cycles of ( 98 c for 10 s and 72 c for 30 s ) ; 72 c for 10 min .
the pcr - amplified oligos were purified using a nucleotide removal kit ( qiagen ) according to manufacturer s recommendations , and subjected to restriction digest in a 70 l reaction containing 1 g dna , 7 l 10x neb buffer 4 , 0.7 l 100x bsa , 40 u of xhoi and mlyi ( 2 l xhoi , 4 l mlyi ) , and incubated in 37 c waterbath for 6 h. digested fragments were verified by electrophoresis on 20% page - tbe with 0.5% tbe running buffer , and purified over a second qiagen nucleotide removal column before cloning into psicor - mcherry . to prepare the vector for cloning , psicormcherry was subject to restriction digest in a 150 l reaction containing 15 l neb buffer 4 , 1.5 l bsa , 5 g psicor - mcherry , 50 u each of xhoi and hpai ( neb ) , and incubated overnight at 37 c .
vector was then treated ( without purification ) with antarctic phosphatase ( neb ) in a 150 l reaction containing 16.9 l antarctic phosphatase buffer and 12.5 u enzyme for 4 h at 37 c .
vector was then purified on an 0.8% agarose gel , cut out , and subject to gel - extraction using the qiaquick gel purification it ( qiagen ) .
vector was then phenol - chloroform extracted , ethanol precipitated , and resuspended in 20 l water .
a second library type was cloned using primers for mir30-context shrnas as previously described4 , 5 , using the above parameters , except cloning was performed by digestion with xhoi and ecori .
ligations were performed in a 20 l reaction containing 500 ng vector , 30 ng insert , 2 l 10x ligase buffer ( neb ) , 2000 u t4 dna ligase ( neb ) , and were incubated at 16 c for 16 h. to preserve the diversity of the library , colonies were scraped from twelve 15 cm plates directly after transformation of the ligation mixture ( six 100 l transformations of max - efficiency dh5a cells ( invitrogen ) ) .
the collected cell pellet was used for maxiprep ( qiagen ) directly , and enough dna was recovered from the plates for numerous viral preps . to select shrnas
that specifically target the human cd45 receptor , lair1/cd305 , or cd3 , we prepared concentrated virus from the cd - antigen library and control ( no insert ) psicor - mcherry vectors as described previously16 . 2  10 human raji b cells , u937 cells , or jurkat cells ( atcc ) were infected with virus at an moi of 0.1 to ensure single integration sites of the viruses .
after 7 days of culture ,  5  10 cells were incubated on ice for 15 min with 500 l of pbs containing 20% normal mouse serum , 5% bsa , and 10% fcs to block non - specific interactions .
subsequently , pe - conjugated anti - cd45 ( bd biosciences ) , apc - conjugated anti - lair1 ( r&d ) , or apc - conjugated anti - cd3 ( bd biosciences ) was added and allowed to interact with the cells for 30 min , followed by two washes with hanks bss medium supplemented with 2% fcs .
mcherry positive ( as a marker for infected cells ) and cd45 or lair-1-reduced cells ( to select potential active anti - cd45 or anti - lair1 shrnas ) were sorted by using a moflow cell sorter ( dako cytomation ) .
after a week of culture the same procedure was repeated , and the cells were cultured for an additional 7 days .
note that for the binning / deep sequencing experiment , cells were infected and sorted after 7 days .
all of the sequences for the cd antigen shrna library as well as the effective shrnas targeting cd45 and lair1 are included as supplementary data 13 online .
to re - test anti - cd45 shrnas following the 2-sort experiment , genomic dna was isolated from the sorted cell fractions , and lentiviral shrna integrations were amplified by pcr using the primers : 5 : tgcaggggaaagaatagtagac , and 3 : agttatgtaacgcggaactcc , cloned into the pcr2.1-topo vector ( invitrogen ) , and sequenced .
identified cd45-targeting shrnas were subcloned into psicor - mcherry , packaged into lentivirus and individually validated for their efficiency in cd45 knock - down , essentially as described above .
analysis of cells after viral infection and surface antigen staining was performed on an lsr ii flow cytometer ( bd biosciences ) and the facs data was analyzed by flowjo software ( tree star , inc . ) .
percent knockdown is calculated by ( ( geo - mean of uninfected cells minus geo - mean of infected cells)/geo - mean of uninfected cells)*100 .
shrnas were amplified from genomic dna in a 50 l pcr reaction consisting of 30 l water , 10 l 5x phusion gc buffer ( finnzymes ) , 5 l 5 m primer mix , 1 l 10 mm dntps , 1.5 l dmso , 750 ng genomic dna , and 1u ( 0.5 l ) phusion polymerase ( finnzymes ) .
cycling parameters were 98 c for 30 s , then 25 cycles of ( 98 c for 30 s , 56 c for 15 s , 72 c for 15 s ) , then 72 c for 10 min . primers for amplification of shrnas from genomic dna were 5 ' ataaatatcccttggagaaaagc , 3 ' ggcggtaatacggttatcca . in some cases
many pcr reactions were pooled on a minelute column ( qiagen ) before electrophoresis on 20% page with 0.5% tbe running buffer , electroelution , and concentration on a second column .
genomic dna was prepared as before , and shrnas were amplified as before , except sequences compatible with annealing to the illumina flow cell were added .
sequencing was performed according to manufacturer s protocols ( illumina ) . for assessing linearity of sequence counting over a dilution series ,
thirty - one 68mer oligos consisting of a unique 28 nt tag flanked by 21 nt and 19 nt common illumina primer binding sites were individually amplified by pcr and purified from an acrylamide gel .
the dsdna products were quantified using a bioanalyzer ( agilent technologies ) , and pooled with a dilution strategy designed to give a broad range of expected sequence tag concentrations .
this pool was subjected to deep sequencing , and reads were aligned with the tag library allowing up to 3 mismatches against the tag .
to design shrnas against cd antigens , we used the shrna prediction algorithm freely available through the hannon lab website : http://katahdin.cshl.org:9331/homepage/portal/scripts/main2.pl15 .
the output shrna sequences were then modified to have a 9-bp loop ( ttcaagaga ) and common primer binding sites compatible with cloning into the lentiviral vector psicor - mcherry which drives stable expression of the hairpin via a mouse u6 promoter16 : 5 ' : cgcctgcgagtctggtatg , 3 : ggaattcgccagctcgag ( reverse complement ) .
a 22,000-element , 96-bp oligonucleotide pilot library was designed using these sequences , and was provided by agilent technologies as a pool in a single tube ( 4 pmol ) and dissolved in 200 l water .
improvements to fidelity of long oligonucleotides were made by minimizing the synthetic cycle loss during coupling and deblock , and by minimizing the depurination side reaction .
we identified optimal conditions for amplification of full - length product ( 96 bp oligos ) by using phusion dna polymerase ( finnzymes ) , varying the amount of dmso , annealing temperature , and extension times for the pcr on a bio - rad icycler .
final conditions were for 50 l reaction : 30 l water , 10 l 5x phusion gc buffer , 1 l 10 mm dntps , 5 l 5 m primer mix , 0.5 l template , 1 l dmso , 0.5 l hot start phusion polymerase ( finnzymes ) .
cycling parameters were 98 c for 30 s ; 15 cycles of ( 98 c for 10 s and 72 c for 30 s ) ; 72 c for 10 min .
the pcr - amplified oligos were purified using a nucleotide removal kit ( qiagen ) according to manufacturer s recommendations , and subjected to restriction digest in a 70 l reaction containing 1 g dna , 7 l 10x neb buffer 4 , 0.7 l 100x bsa , 40 u of xhoi and mlyi ( 2 l xhoi , 4 l mlyi ) , and incubated in 37 c waterbath for 6 h. digested fragments were verified by electrophoresis on 20% page - tbe with 0.5% tbe running buffer , and purified over a second qiagen nucleotide removal column before cloning into psicor - mcherry . to prepare the vector for cloning , psicormcherry was subject to restriction digest in a 150 l reaction containing 15 l neb buffer 4 , 1.5 l bsa , 5 g psicor - mcherry , 50 u each of xhoi and hpai ( neb ) , and incubated overnight at 37 c .
vector was then treated ( without purification ) with antarctic phosphatase ( neb ) in a 150 l reaction containing 16.9 l antarctic phosphatase buffer and 12.5 u enzyme for 4 h at 37 c .
vector was then purified on an 0.8% agarose gel , cut out , and subject to gel - extraction using the qiaquick gel purification it ( qiagen ) .
vector was then phenol - chloroform extracted , ethanol precipitated , and resuspended in 20 l water .
a second library type was cloned using primers for mir30-context shrnas as previously described4 , 5 , using the above parameters , except cloning was performed by digestion with xhoi and ecori .
ligations were performed in a 20 l reaction containing 500 ng vector , 30 ng insert , 2 l 10x ligase buffer ( neb ) , 2000 u t4 dna ligase ( neb ) , and were incubated at 16 c for 16 h. to preserve the diversity of the library , colonies were scraped from twelve 15 cm plates directly after transformation of the ligation mixture ( six 100 l transformations of max - efficiency dh5a cells ( invitrogen ) ) .
the collected cell pellet was used for maxiprep ( qiagen ) directly , and enough dna was recovered from the plates for numerous viral preps .
to select shrnas that specifically target the human cd45 receptor , lair1/cd305 , or cd3 , we prepared concentrated virus from the cd - antigen library and control ( no insert ) psicor - mcherry vectors as described previously16 .
2  10 human raji b cells , u937 cells , or jurkat cells ( atcc ) were infected with virus at an moi of 0.1 to ensure single integration sites of the viruses .
after 7 days of culture ,  5  10 cells were incubated on ice for 15 min with 500 l of pbs containing 20% normal mouse serum , 5% bsa , and 10% fcs to block non - specific interactions .
subsequently , pe - conjugated anti - cd45 ( bd biosciences ) , apc - conjugated anti - lair1 ( r&d ) , or apc - conjugated anti - cd3 ( bd biosciences ) was added and allowed to interact with the cells for 30 min , followed by two washes with hanks bss medium supplemented with 2% fcs .
mcherry positive ( as a marker for infected cells ) and cd45 or lair-1-reduced cells ( to select potential active anti - cd45 or anti - lair1 shrnas ) were sorted by using a moflow cell sorter ( dako cytomation ) . after a week of culture the same procedure
note that for the binning / deep sequencing experiment , cells were infected and sorted after 7 days .
all of the sequences for the cd antigen shrna library as well as the effective shrnas targeting cd45 and lair1 are included as supplementary data 13 online .
to re - test anti - cd45 shrnas following the 2-sort experiment , genomic dna was isolated from the sorted cell fractions , and lentiviral shrna integrations were amplified by pcr using the primers : 5 : tgcaggggaaagaatagtagac , and 3 : agttatgtaacgcggaactcc , cloned into the pcr2.1-topo vector ( invitrogen ) , and sequenced .
identified cd45-targeting shrnas were subcloned into psicor - mcherry , packaged into lentivirus and individually validated for their efficiency in cd45 knock - down , essentially as described above .
analysis of cells after viral infection and surface antigen staining was performed on an lsr ii flow cytometer ( bd biosciences ) and the facs data was analyzed by flowjo software ( tree star , inc . ) .
percent knockdown is calculated by ( ( geo - mean of uninfected cells minus geo - mean of infected cells)/geo - mean of uninfected cells)*100 .
shrnas were amplified from genomic dna in a 50 l pcr reaction consisting of 30 l water , 10 l 5x phusion gc buffer ( finnzymes ) , 5 l 5 m primer mix , 1 l 10 mm dntps , 1.5 l dmso , 750 ng genomic dna , and 1u ( 0.5 l ) phusion polymerase ( finnzymes ) .
cycling parameters were 98 c for 30 s , then 25 cycles of ( 98 c for 30 s , 56 c for 15 s , 72 c for 15 s ) , then 72 c for 10 min . primers for amplification of shrnas from genomic dna were 5 ' ataaatatcccttggagaaaagc , 3 ' ggcggtaatacggttatcca . in some cases
many pcr reactions were pooled on a minelute column ( qiagen ) before electrophoresis on 20% page with 0.5% tbe running buffer , electroelution , and concentration on a second column .
genomic dna was prepared as before , and shrnas were amplified as before , except sequences compatible with annealing to the illumina flow cell were added .
final primer sequences were : 5 : aatgatacggcgaccaccgacactctttccctcccttggagaaaagccttgtttg and 3 : caagcagaagacggcatacga atggatccta gtactcgag . note that parts of these oligonucleotide sequences are copyrighted by illumina , inc . the sequencing primer used was cactctttccctcccttggagaaaagccttgtttg .
sequencing was performed according to manufacturer s protocols ( illumina ) . for assessing linearity of sequence counting over a dilution series ,
thirty - one 68mer oligos consisting of a unique 28 nt tag flanked by 21 nt and 19 nt common illumina primer binding sites were individually amplified by pcr and purified from an acrylamide gel .
the dsdna products were quantified using a bioanalyzer ( agilent technologies ) , and pooled with a dilution strategy designed to give a broad range of expected sequence tag concentrations .
this pool was subjected to deep sequencing , and reads were aligned with the tag library allowing up to 3 mismatches against the tag .
 short hairpin rna ( shrna ) libraries are limited by the low efficacy of many shrnas , giving false negatives , and off - target effects , giving false positives . here we present a strategy for rapidly creating expanded shrna pools ( 30 shrnas / gene ) that are analyzed by deep - sequencing ( expand ) . 
 this approach enables identification of multiple effective target - specific shrnas from a complex pool , allowing a rigorous statistical evaluation of whether a gene is a true hit . 
Methods
Amplification, digestion, cloning, and preparation of shRNA library
Isolation of CD45, LAIR1, or CD3-targeting shRNAs
Amplification of shRNA pools from genomic DNA
Deep Sequencing, and Quantitation of Read Accuracy
Supplementary Material