title: GSM8141789: Knockout, tdTomato+ MBC, D7, rep3; Mus musculus; RNA-Seq
library_name: GSM8141789
library_construction_protocol: 5 mice were used for cell-sorting in each experiment. Splenocyte single cell suspensions were generated by crushing spleens in MACS buffer (PBS supplemented with 1% FBS and 5 mM EDTA). Cells were filtered through 70 uM nylon mesh and washed at 1700 RPM, 4 ⁰C, 5 minutes. Pellets were resuspended in RT ACK lysis buffer and incubated RT for 3 minutes, followed by addition of 5-10 volumes of MACS buffer and washing at 1700 RPM, 4 ⁰C, 5 minutes. Single-cell suspension of splenocytes was stained in MACS buffer containing Fc blocker (supernatant from 2.4G2 cell culture) for 20 min on ice before surface staining with primary antibodies in appropriate concentrations on ice for 25 min. Cells were washed again with 5-10 volumes of MACS, and resuspended in MACS for analysis on BD Aria II. Approximately 200 cells of desired type were sorted into 6 μl lysis buffer (0.2% Triton X-100 and 2 U/μl of RNase inhibitor). Lysates were mixed with 3 μl of anchored oligo-dT primer (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and 3 μl of dNTP mix (10 mM, Fermentas), denatured at 72 °C for 3 min and immediately placed on ice afterwards. The first-strand reaction mix containing 1 μl SuperScript II reverse transcriptase (Invitrogen), 0.5 μl RNAse inhibitor (Clontech), 4 μl Superscript II First-Strand Buffer (Invitrogen), 1 μl DTT (100 mM, Invitrogen), 4 μl betaine (5 M, Sigma), 0.12 μl MgCl2 (1 M, Sigma), 0.2 μl TSO (100 μM) and 0.58 μl nuclease-free water, were added to each sample. Reverse transcription reaction was carried out by incubating at 42 °C for 90 min, followed by 10 cycles of (50 °C for 2 min, 42 °C for 2 min). Finally, the reverse transcriptase was inactivated by incubation at 70 °C for 15 min. PCR mix containing 25 μl KAPA HiFi HotStart ReadyMix, 0.5 μl IS PCR primers (10 μM) and 1.1 μl nuclease-free water were added to each sample. The reaction was incubated at 98 °C for 3 min, then cycled 17 times (98 °C 20 s, 67 °C 15 s, 72 °C 6 min), with a final extension at 72 °C for 5 min. cDNAs were purified by Vazyme DNA clean beads following manufacturer's instructions. 1 ng of cDNA were used for the tagmentation reaction using Tn5 (Vazyme). The tagmentation reaction (20 μl) was incubated at 55 °C for 10 min and stopped by addition of 2 μl 0.2%SDS and incubated at 70 °C for 20 min. A second amplification was performed using Q5 polymerase (NEB) as follows: 72 °C 3 min, 98 °C 30 s, then 12 cycles of (98 °C 15 s, 60 °C 30 s, 72 °C 3 min), final extension at 72 °C 5 min. Libraries were purified using Vazyme DNA clean beads.