title: GSM6923355: CTL EXO 02; Homo sapiens; miRNA-Seq
library_construction_protocol: Use the 200ng-1μg of RNA sample，then separate RNA segment of different size by PAGE gel, select 18-30nt(14-30 ssRNA Ladder Marker，TAKARA)stripe and recycle; Adaptor ligation:Prepare connection 3'adaptor system (Reaction condition:70°C for 2min; 25°C for 2h);Secondly add RT-Primer, (Reaction condition: 65°C for 15min;ramp to 4°C at a rate of 0.3°C/sec);Thirdly add 5'adaptor mix system(Reaction condition:70°C for 2min; 25°C for 1h);  RT -PCR:Prepare First Strand Master Mix and Super Script II (Invitrogen) reverse transcription (Reaction condition:42°C for 1h;70°C for 15min);Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments (Reaction condition:95°C for 3min;15-18 cycles of(98°C for 20s，56°C for 15s，72°C for 15s);72°C for 10min;4°C hold) ;  Purify PCR products:Then the PCR products were purified with PAGE gel，dissolve the recylced products in EB solution.  2.3 Circularization  The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library.  2.4 Library quality control  Library was validating on the Agilent Technologies 2100 bioanalyzer.