title: GSM2445409: GBM4_Cell_smallRNA; Homo sapiens; RNA-Seq
library_construction_protocol: Approximate 100 mL conditioned media was used as input for exRNA isolation. Conditioned media was centrifuged at 300 g, 4 °C for 10 min, following by the additional centrifugation at 2,000 g, 4 °C for 15 min, to remove cells and cell debris. Five uL of SUPERase In RNase Inhibitor (Ambion) was added to the supernatants per 10 mL media. The media was then filtered sequentially through the 2 um filter (GE Healthcare, UK), 0.8 um filter (EMD Millipore, MA), and 0.22 um filter (EMD Millipore), with no/minimal pressure applied. The filtrate was split to 15 mL per sample and further filtered through the 0.02 um filter (GE Healthcare) with up to 75 psi pressure applied. To facilitate the 0.02 um filtration, a mechanical syringe pump was designed and manufactured (Fig. S2). Upon filtration, each filter was washed with 1 mL DPBS (Corning), and the corresponding fractions were lysed with 600 uL Lysis Solution of the miRCURY RNA Isolation Kit – Cell & Plant (Exiqon, Denmark). The fractions collected on 0.02 um filters were lysed with 900 uL Lysis Solution. The last flow-through fractions of 0.02 um filters were pooled together (up to 30mL) and concentrated approximately 60 times using 3 kDa Amicon Ultra Centrifugal Filters (EMD Millipore) at 4,000 g, 4 °C, for 60 min. The concentrates were collected and lysed with six volumes of the same Lysis Solution (Exiqon). Total RNA was then isolated from all fractions as recommended by miRCURY protocol, with on-column DNase treatment (Qiagen, Germany). The corresponding 1.2 mL of the source neurospheres were span down at 300 g, 4 °C for 5 min, and total cellular RNA was isolated from them and analyzed in parallel. The same protocol was carried out for RNA isolation from fresh media, with 500 mL media input. For RNA isolation from primary cells cultured in 24-wells plates, the cells were lysed with 350 uL Lysis Solution per well. The concentrations of cellular and extracellular RNA were determined using the NanoDrop 2000 Spectrophotometer and Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific), respectively. The RNA quality was examined using Agilent 2100 Bioanalyzer (Agilent, CA) and the RNA Integrity Number (RIN) estimated. Two sets of spike-in RNAs were added to the samples prior to library preparation: ERCC RNA Spike-In Control Mixes (Ambion; 0.02 uL per one ug total RNA), and miRCURY Spike-in kit, part 1, with UniSp2's final concentrations of 1.25 and 5.0 fmol per one ug of total RNA for cellular and extracellular RNA, respectively. Total RNA, either 40~200 ng of exRNA, or 2 ug of cellular RNA, was rRNA-depleted using the Ribo-Zero rRNA Removal Kits (Illumina, CA). One quarter of the rRNA-depleted RNA was fragmented to 100~500 nt using the 5X First-Strand Buffer (Clontech, CA), and utilized for the long RNA library construction by SMARTer Stranded RNA-Seq Kit (Clontech). The remaining 75% of the rRNA-depleted RNA was treated sequentially with Tobacco Acid Pyrophosphatase (TAP; Illumina) and T4 Polynucleotide Kinase (T4PNK; New England Biolabs, MA) to create more uniform 5'- and 3'- ends for various classes of transcripts. The RNA was then used as input for the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs), with size selection of 15-65nt inserts for small RNA libraries. The quality of libraries was examined using the Agilent DNA 1000 kit at the Agilent 2100 Bioanalyzer instrument, and cDNA quantified by qRT-PCR with TaqMan Probe (Thermo Fisher Scientific). The libraries were sequenced on HiSeq 2000 (Illumina) with single read 50 cycles by Beijing Genomics Institute (BGI, China).