GSM4981230: sncRNA organ atlas, Homo sapiens, spleen_4; Homo sapiens; ncRNA-Seq
Total RNA including smallRNAs was isolated using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's recommendations. In brief, tissues were lysed and homogenized in 700 µl QIAzol Lysis Reagent using 5 mm stainless steel beads and the TissueLyser LT (Qiagen) for 5 min at 50 Hz. After 5 min incubation, samples were mixed with 140 µl chloroform and phase separation was performed at 12000 x g and 4 °C for 15 min. The RNA in the aqueous phase was precipitated by adding 1.5 Volume of 100 % ethanol and purified using the RNeasy columns provided with the kit, either manually or semi-automated using the Qiacube instrument with the respective protocols provided for the miRNeasy Mini kit. We performed library preparation for sequencing on the BGISEQ-500RS following the instructions of the MGIEasy Small RNA Library Prep Kit (MGI Technologies). To limit the bias the manual library preparation performed previously was replaced by a semi-automated workflow on the MGI-SP100. In general, 100 ng total RNA was used as input material for the library preparation. The library construction includes of 3' adapter ligation, 3' adapter digestion, 5' adapter ligation followed by reverse transcription (RT) of RNA into cDNA using RT primers which contained specific barcodes. Next, cDNA was amplified by PCR in 22 cycles. The PCR products were separated using 6 % TBE gels and bands of the size around 110 bp were cut from the gel.  After purification, the DNA yield was measured with the Qubit® 1xdsDNA HS Assay Kit (Invitrogen). A total of 16 different barcoded samples were pooled together in equal amounts and circularized to generate a single-stranded DNA library. The BGISEQ-500RS uses DNA nanoballs (DNBs) for sequencing, which were generated by rolling circle amplification (RCA). DNBs were then loaded into the patterned nanoarray using the BGIDL-50 DNB loader. Finally, single-end sequencing was performed on the BGISEQ-500RS using the High-throughput Sequencing Set (SE50) (Small RNA) according to the manufacturer's instructions. The mouse samples have been processed with an advanced pipeline using the higher-throughput SP-960 sample prep system and MGISEQ-400 sequencing system. In the latter case we made further use of the CoolMPS sequencing chemistry.