GSM1876151: Pre-TFH cells [CC3]; Mus musculus; miRNA-Seq
RNA was harvested using Trizol reagent. Approximately 1 µg of total RNA was used for Next Generation Sequencing following the standard Illumina protocol, TruSeq_SmallRNA_SamplePrep_Guide_15004197_C.pdf, using 12 cycles of PCR. 3' Adapter Ligation. 10 pmol of 3'-adenylated DNA adapter (5'-AppTGGAATTCTCGGGTGCCAAGGddC) is combined with 1 ug total RNA in a 4 ul volume, heated to 70 degrees C for 2 minutes and then placed on ice.  In a separate tube, 2 uL 10X T4 RNA Ligase 2 Truncated reaction buffer(NEB), 2 uL 50% PEG8000 (NEB), 1 uL RNase OUT (Lifetech) and 1 uL T4 RNA Ligase 2 Truncated enzyme (NEB) were combined and added to the RNA/3'adapter mix and incubated at 28 degrees C for one hour. This mixture was then heated to 70 degrees C for 2 minutes.  5' Adapter Ligation. Next, 1 uL water, 1 uL 10 mM ATP, 1 uL T4 RNA Ligase and 1 uL 5' RNA adapter (25 pmol, 5'-GUUCAGAGUUCUACAGUCCGACGAUC) were added and incubated at 28°C for 1 hour.  Reverse Transcription. Next, 1 uL of a 10 uM solution of RT primer (5'-GCCTTGGCACCCGAGAATTCCA) was added to 6 uL of the 5'adapter ligation mix above heated to 70 degrees C for 2 minutes and then transferred to ice. This was then combined with a solution containing: 2 uL of 5X First Strand Buffer (Superscript II buffer, Lifetech), 0.5 uL of 12.5 mM dNTPs, 1 uL 0.1 M DTT, 1 uL RNaseOUT (Lifetech), and 1 uL Superscript II (200 U/uL). The reaction was incubated at 50 degrees C for 1 hour and then placed on ice. PCR. The 12.5 uL reverse transcription reaction above was then added to a tube containing: 30 pmol of Illumina Truseq Universal PCR primer (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA); 30 pmol of Illumina Truseq Barcoded PCR primer (5'-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA, where XXXXXX, correspond to standard Illumina Truseq barcodes); and 25 uL of 2X KAPA HiFi HotStart ReadyMix PCR Kit in a 50 uL total volume. The reaction was heated to 98 degrees C for 1 minute, and then cycled 12 times with 98 degrees C for 10 seconds, 60 degrees C for 30 seconds, and 72 degrees C for 15 seconds, followed by a single incubation at 72 degrees C for 5 minutes. The resulting PCR products were isolated using AmpureXp magnetic bead purification (Agencourt, Beckman-Coulter) and then purified on 4% Agarose gel.
GEO Accession
GSM1876151