title: GSM3671700: EXR-MBITZ1CTOT1SAMPB1A8-BS: CTOT04_01_Urine_1-8; Homo sapiens; ncRNA-Seq
library_construction_protocol: 450 ul biofluid sample were mixed with 105 ul buffer P, digested with 28 ul Proteinase K working solution at 65C for 8 minutes, extracted with 517 ul ED2 working solution, cooled to 10C, phase separation was induced after adding 110 ul chloroform, approx. 600 ul aqueous supernatants were mixed with 1200 ul buffer VB2G, loaded on Zymo-I 96-well silica matrix plate, washed twice with 970 ul EWL, once with 970 ul 100% ethanol, twice with 500 ul 80% ethanol and eluted into 20 ul TrisHCl pH 7.5. Protocol was executed on epMotion 5075 liquid handling system. Low speed centrifugation (1200 g x 10 minutes) was performed for cell removal. No Kit was used. Multiplexed Small RNA library preparation protocol developed by Tuschl lab. Adenylated DNA adapters each containing a unique 5-nt barcode sequence were ligated to the 3end of the RNA overnight at 4C.The barcoded adapters allow 24 individual RNA samples to be pooled into one lane.The pooled samples were purified on a 15% polyacrylamide gel and size-selection was guided by radiolabeled RNA markers corresponding to a sRNA size of 19-30 nt. Next, a 26-nt RNA adapter compatible with Illumina sequencing was ligated to the 5end of the RNA at 37C for 1 hour and samples were purified on a 12% polyacrylamide gel.  The cDNA libraries were then generated by reverse transcription and amplified using up to 20 PCR cycles, and the final libraries ranging from 90-101 nt were purified on a 2.5% agarose gel and sequenced in a single Illumina HiSeq lane.
attributes.tag: GEO Accession
attributes.value: GSM3671700